Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC fGHZFhVJjBWkTZwrTqZZzBw S0066-782X2021005018202 S0066-782X2021001401134 abc.20200672 10.36660/abc.20200672 Artigo Original O Pré-Condicionamento com Dexmedetomidina Reduz a Lesão de Isquemia-Reperfusão do Miocárdio em Ratos, Inibindo a Via PERK Chen YuJiao 1 2 Cao Song 1 0000-0002-0707-7671 Chen Hui 1 Yin CunZhi 1 Xu XinPeng 1 Yang ZaiQun 1 Zunyi Medical University Zunyi Guizhou China Zunyi Medical University, Zunyi, Guizhou – China Affiliated Hospital North Sichuan Medical College NanChong SiChuan China Affiliated Hospital of North Sichuan Medical College, NanChong, SiChuan - China Correspondência: Hui Chen • Zunyi Medical College Affiliated Hospital - 149 Dalian road, Zunyi, Guizhou Zunyi 563003 – China E-mail: 2267390140@qq.com

Contribuição dos autores

Concepção e desenho da pesquisa: Chen Y, Chen H; Obtenção de dados: Chen Y, Cao S, Chen H, Yin C, Xu X, Yang Z; Análise e interpretação dos dados: Chen Y, Yang Z; Análise estatística: Chen Y, Cao S, Yin C; Obtenção de financiamento e Redação do manuscrito: Chen Y; Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Chen Y, Xu X.

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 08 10 2021 12 2021 117 6 1134 1144 12 07 2020 27 10 2020 27 01 2021 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Resumo Fundamento

A cardiopatia isquêmica atraiu muito atenção devido às altas taxas de mortalidade, custos do tratamento e a crescente morbidade na população jovem. Estratégias de reperfusão reduziram a mortalidade. Porém, a reperfusão pode levar à morte do cardiomiócito e subsequente dano irreversível ao miocárdio. No momento, não há um tratamento eficiente e direcionado para a lesão de isquemia-reperfusão (I/R).

 Objetivos

Avaliar se a dexmedetomidina (DEX) tem efeito protetivo na I/R do miocárdio e explorar os possíveis mecanismos por trás dela.

 Métodos

Corações de ratos foram perfundidos com o sistema de perfusão de Langendorff e aleatoriamente distribuídos em cinco grupos: grupo controle, perfundido com solução de Krebs-Henseleit (K-H) por 205 minutos sem isquemia; e quatro grupos de teste que foram submetidos a 40 minutos de isquemia global e 120 minutos de reperfusão. O Grupo DEX, o grupo ioimbina (IO) e o grupo DEX + IO foram perfundidos com DEX (10 nM), IO (1 μM) ou a combinação de DEX e IO antes da reperfusão, respectivamente. A hemodinâmica cardíaca, o tamanho do infarto do miocárdio e a histologia do miocárdio foram avaliados. A expressão da proteína-78 regulada pela glicose (GRP78), a proteína quinase do retículo endoplasmático (PERK), a PERK fosforilada, o fator de iniciação eucariótico 2α (eIF2α), eIF2α fosforilado, o fator de transcrição 4 (TCF-4) e a proteína homóloga à proteína ligadora do acentuador CCAAT (CHOP) foram avaliados. P< 0,05 foi considerado para indicar a diferença estatisticamente significativa.

 Resultados

O pré-condicionamento com DEX melhorou a função cardíaca nos corações com I/R, reduziu o infarto do miocárdio, a apoptose do miocárdio e a expressão de GRP78, p-PERK, eIF2α, p-eIF2α, TCF-4 e CHOP.

 Conclusões

O pré-tratamento com DEX reduziu a lesão de I/R no miocárdio ao suprimir a apoptose, o que foi induzido pela via PERK.

 Dexmedetomidina cardiopatia Isquêmica mortalidade Reperfusão Miocárdica Pre-Condicionamento Hipnoticos e Sedativos Proteínas Quinases Ratos The Science and Technology Development Plan of Guizhou Province 2016[1175] The Postgraduate Research Fund of Guizhou Province KYJJ2017034 Fontes de financiamento: O presente estudo foi financiado por The Science and Technology Development Plan of Guizhou Province (grant no.2016[1175]), and The Postgraduate Research Fund of Guizhou Province (grant no. KYJJ2017034). Introdução

A cardiopatia isquêmica tem atraído muita atenção devido às suas altas taxas de mortalidade, custos do tratamento e a crescente morbidade na população jovem. Nas últimas três décadas, estratégias de reperfusão para aliviar a lesão isquêmica têm sido incluídas na prática clínica. Porém, a reperfusão pode levar à morte do cardiomiócito e subsequente dano irreversível ao miocárdio.1

A lesão de isquemia-reperfusão do miocárdio é um processo fisiopatológico de longo prazo e complexo. O processo de restabelecer o fluxo sanguíneo ao miocárdio isquêmico pode levar a uma grande variedade de mudanças biológicas básicas, incluindo deficiência na homeostase iônica, ruptura de radicais de oxigênio, autofagia, disfunções metabólicas do trifosfato do ATP, estresse oxidativo, disfunção mitocondrial e apoptose.2 Estudos recentes demonstraram que o dano causado ao miocárdio envolve dois processos: lesão de isquemia-repercussão (I/R). A lesão de I/R é um grande determinante da mortalidade de longo prazo; por isso, a possibilidade de aliviar a extensão da lesão tem grande valor individual e socioeconômico.3 - 7

Foi demonstrado que a apoptose do cardiomiócito é um importante mecanismo da lesão de I/R do miocárdio.8 A apoptose é acionada pelo receptor mitocondrial e da morte nas condições da reperfusão miocárdica.9 Porém, esses mecanismos ainda precisam ser determinados.2 Estudos recentes exploraram a associação entre o retículo endoplasmático (RE) e a lesão de I/R do miocárdio.9 - 11 Condições de estresse, como I/R, hipóxia e estresse oxidativo foram identificados na desregulação das funções do RE, levando ao estresse do RE.12 As consequências patológicas da ruptura do lúmen do RE e da falta de comunicação foram implicadas na lesão de I/R do miocárdio.13 , 14 Porém, não está claro se o estresse do RE induz a apoptose ao ativar a PERK durante a lesão de reperfusão do miocárdio.15

No momento, devido à falta de medicamentos específicos e terapias padronizadas, não há tratamento preciso e direcionado para a lesão de I/R. A dexmedetomidina (DEX) é agonista do receptor α2 adrenérgico (RA), e tem impacto limitado na hemodinâmica e na respiração; por isso, promove a sedação e analgesia ideais para pacientes submetidos à cirurgia cardiovascular.16 Estudos anteriores demonstraram que o pré-condicionamento com DEX trouxe efeitos cardioprotetores em corações isquêmicos.17 - 20 Porém, o mecanismo molecular da proteção da DEX, até hoje, é desconhecido. Este estudo avaliou a hipótese de que o pré-tratamento com DEX protege o coração da lesão de I/R por meio da ativação da PERK, o que depende da ativação do receptor α2. Assim, este estudo usou um modelo de coração isolado com I/R para avaliar os efeitos da DEX na lesão da I/R e seu potencial mecanismo.

 Métodos Experimento com animais

Ratos machos e adultos da linhagem Sprague-Dawley (8 a 10 semanas de idade; 260-280g) foram obtidos no centro de experimento com animais de Changsha (Changsha, China). Todos os procedimentos experimentais estavam de acordo com o “Guia para o Cuidado e Uso de Animais de Laboratório”, publicado na China (n. 1492, 2001), e foram aprovados pelo Comitê de Cuidado e Uso de Animais Experimentais da Zunyi Medical University. Todos os ratos foram acomodados em condições padrão (temperatura ambiente, 22ºC, ciclo claro/escuro de 12 horas), com acesso livre à comida e à água, no laboratório Guizhou Key Laboratory of Anesthesiology and Organ Protection, da Zunyi Medical University.

 Isolamento dos corações dos ratos adultos

Os ratos foram anestesiados com pentobarbital sódico (45 mg/kg, via intraperitoneal). Depois do sucesso da anestesia, o coração foi imediatamente retirado do peito por meio da esternotomia e imerso em solução gelada de Krebs-Henseleit (K-H) (NaCl, 119 mM; KCl, 6,0 mM; CaCl2, 1,24 mM; NaHCO3, 20,1 mM; KH2PO4, 1,24 mM; MgSO4, 1,24 mM; glicose, 11,2 mM). O coração foi perfundido de modo retrógrado usando o método de Langendorff pela aorta (PanLab). A solução de K-H foi perfundida sob constante pressão de perfusão de 75-80 mmHg, e balanceada com uma mistura de 95% de O2 e 5% de CO2, com pH de 7,35-7,45 a 37°C. Um balão de látex conectado a um transdutor de pressão foi inserido no ventrículo esquerdo por meio da válvula mitral, e preenchido com soro fisiológico para produzir pressão diastólica final do ventrículo esquerdo (PDFVE) de 2-10 mmHg. O volume do balão foi mantido constante durante o experimento. A frequência cardíaca (FR), a PDFVE, a pressão desenvolvida no ventrículo esquerdo (PDVE) (PDVE = pressão sistólica no ventrículo esquerdo (PSVE) – PDFVE), a pressão ventricular esquerda máxima de mudanças positivas e negativas (±dp/dtmax) e o produto de taxa de pressão (PTP = FC x PDVE) foram registrados pelo sistema Lab Chart.

 I/R e pré-condicionamento com DEX em corações isolados de ratos

Para cada teste, após balancear a perfusão por 15 minutos, os corações cuja PDVE de base e FC eram >50mmHg e >200 batidas/min foram aleatoriamente distribuídos em cinco grupos, em envelopes lacrados. As amostras em todos os grupos foram perfundidas por 30 minutos antes dos 40 minutos de isquemia global normotérmica, seguido de 120 minutos de reperfusão, exceto no grupo controle (n=6): (i) grupo controle, sem protocolos de pré-condicionamento, perfundidos com solução de K-H normal; (ii), grupo de I/R, sem protocolos de pré-condicionamento, perfundidos com solução de K-H normal antes da isquemia; (iii) grupo DEX, pré-condicionado com 10 nM de DEX antes da isquemia; (iv) grupo ioimbina (IO), pré-condicionado com 1 μM IO antes da isquemia; e (v) grupo DEX + IO, pré-condicionado com 10 nM DEX + 1 μM IO antes da isquemia. As concentrações de DEX (Hengrui pharmaceutical) e do antagonista foram selecionadas com base em estudos anteriores. A ioimbina (IO) foi usada como antagonista do receptor α2 adrenérgico. DEX e IO foram dissolvidas em solução modificada de Krebs-Ringer ( Figura 1 ).

– <italic>Modelo de Langendorff da lesão de isquemia-reperfusão do miocárdio. Para cada teste, após 15 minutos de balanceamento da perfusão, os corações preparados foram aleatoriamente distribuídos em cinco grupos para igualar a PDVE e a FC, que estavam a menos de 50 mmHg e 200 batidas/min. As amostras no grupo controle eram continuamente perfundidas com solução de K-H por 205 minutos. As amostras nos outros quatro grupos foram perfundidas por 30 minutos antes dos 40 minutos da isquemia global normotérmica, seguidos de 120 minutos de reperfusão. As amostras no grupo I/R não tiveram protocolo de pré-condicionamento e foram perfundidas com solução normal de K-H antes da isquemia; as amostras no grupo DEX foram perfundidas com DEX (10 nM) antes da isquemia; as amostras no grupo IO foram perfundidas com IO (1 μM) antes da isquemia; as amostras do grupo DEX + IO foram perfundidas com DEX (10 nM)+IO (1 μM) antes da isquemia. PDVE: pressão desenvolvida no ventrículo esquerdo, FC: frequência cardíaca, K-H: Krebs-Henseleit, I/R: isquemia-reperfusão, DEX: dexmedetomidina, IO: ioimbina</italic>

Determinação do tamanho do infarto

O tamanho do infarto foi avaliado com a coloração do cloreto de trifeniltetrazólio (TTC). Depois da reperfusão, os tecidos que aderiram à raiz arterial foram removidos, e todo o coração foi congelado. O coração congelado foi cortado em fatias de 1-2 mm. As fatias foram imersas em um frasco com 1% de TTC e incubadas em banho-maria por 30 minutos, a 37ºC. O frasco era continuamente agitado para obter a coloração equilibrada. Então, as fatias foram colocadas em formaldeído 10% por 24 horas, transferidas para uma placa de vidro e uma tampa de vidro foi colocada sobre as fatias. Calços de 2 mm foram colocados nas laterais, entre os vidros, para obter a grossura da fatia, e foram tiradas fotografias digitais. As áreas do infarto e as áreas normais foram determinadas de forma cega, por meio da planimetria, com o software Image-Pro Plus 6.0 (Media Cybernetics, Inc.), e o tamanho do infarto foi demonstrado como um percentual da área total.

 Exame histopatológico

Os tecidos do miocárdio coletados dos ratos foram cortados em seções de 1 cm, fixados em paraformaldeído 4% e embebidos em parafina. Para quantificar a extensão do dano ao miocárdio, as biópsias foram recortadas em seções de 5 µm, que foram coradas com hematoxilina e eosina (HE) por 90 minutos, em temperatura ambiente, e seis campos de visão (ampliação, x200) foram selecionados aleatoriamente para avaliação em cada grupo.

 Método TUNEL

O método de marcação de terminações dUTP e deoxinucleotidil terminal transferase (TUNEL) foi realizado para determinar a extensão da apoptose no miocárdio utilizando um kit comercializado (Baiao Cisco Biological Technology), de acordo com as instruções do fabricante. A marcação nuclear verde foi definida para células TUNEL positivas. Para determinar a extensão da apoptose do miocárdio, cinco campos (ampliação, x200) foram selecionados aleatoriamente de duas seções em cada grupo, e o índice apoptótico (IA) foi calculado por meio do software Image-Pro Plus 6.0 (AI): número de células apoptóticas / número total de células contadas.

 Western blot

O coração foi homogeneizado e lisado com uma solução tampão RIPA (Thermo Fisher scientific, Inc). Os tecidos foram centrifugados a 125 x g por seis minutos, e o sobrenadante foi coletado. A concentração da proteína foi determinada pelo método BCA (Thermo Fisher scientific, Inc.). As amostras foram separadas em eletroforese em gel de poliacrilamida-dodecil sulfato de sódio 10% e transferidas para membranas de nitrocelulose. As membranas foram bloqueadas com leite desnatado por duas horas. As membranas foram analisadas com os anticorpos primários anti-GRP78 (1:1000; cat n. PA1-014A; Thermo Fisher scientific), anti-PERK (1:1000; cat n. PA5-79193; Thermo Fisher scientific), anti-p-PERK (1:1000; cat n. MA5-15033; Thermo Fisher scientific), anti-eIF2α (1:1000; cat n. MA1-079; Thermo Fisher scientific), anti-p-eIF2α (1:1000; cat n. 44-728G; Thermo Fisher scientific), anti-TCF-4 (1:1000; cat n. PA5-68802; Thermo Fisher scientific), anti-CHOP (1:1000; cat n. PA5-86145; Thermo Fisher scientific), anti-GAPDH (1:1000; cat n. MA5-32539; Thermo Fisher scientific) a 4ºC durante a noite. Depois de lavar as membranas três vezes, as amostras foram incubadas com anticorpos secundários conjugados com peroxidase de rábano (1:1000; cat n. G-21234; Thermo Fisher scientific) por duas horas. O conteúdo da proteína foi determinado pelo sistema de imagem em gel (Tanon Science & Technology).

 Análise estatística

O tamanho da amostra do estudo foi baseado em um experimento preliminar. As médias de PDFVE no grupo controle, no grupo DEX e no grupo de I/R, seguido da reperfusão por 120 minutos foram 12,52; 16,23; 22,39, respectivamente, e o desvio padrão foi de 1,45; 2,44 e 145, respectivamente. O poder (1 – β) foi considerado como 0,8. Para os grupos controle e DEX, o tamanho da amostra era seis para cada grupo. Para os grupos I/R e DEX, a amostra era composta de quatro em cada grupo. Então, o tamanho de amostra de seis foi determinado por grupo. Os dados são apresentados como média ± desvio padrão (DP). O SPSS 20.0 (IBM Corp) foi utilizado para a análise estatística. As diferenças entre os grupos foram analisadas com a análise de variância (ANOVA). O teste de Shapiro-Wilk foi utilizado para verificar a normalidade. O teste de diferença mínima (LSD) foi usado para comparar a homogeneidade da variância, e o teste T3 de Dunnett foi aplicado para comparar a variância heterogênea. P < 0,05 foi considerado para indicar a diferença estatisticamente significante.

 Resultados DEX melhora a recuperação cardíaca da lesão de I/R

Para determinar os efeitos da DEX na lesão de I/R do miocárdio, a preparação isolada do coração perfundido foi realizada ao perfundir corações de ratos por 30 minutos, com solução modificada de K-H, antes de 40 minutos de isquemia global, seguidos de 120 minutos de reperfusão. A análise de variância demonstrou que não houve diferenças significativas na hemodinâmica entre os grupos ao final do ponto de equilíbrio. Depois de 45 minutos de reperfusão, não houve diferenças significativas na FC, ±dp/dtmax, PDVE, PDFVE e PTP, exceto no grupo IO e no grupo DEX + IO. No grupo controle, FC, ±dp/dtmax, PDVE e PTP foram maiores, e a PDFVE foi menor em comparação aos outros grupos após a reperfusão por 120 minutos. Isso sugere que a isquemia de 40 minutos em temperatura ambiente foi desafiadora para os corações dos ratos. Durante a segunda metade da reperfusão de 120 minutos, muitos corações exibiram falência e arritmia (~10%). Porém, no grupo DEX, FC, ±dp/dtmax, PDVE e PTP ficaram maiores, e a PDFVE ficou menor em comparação aos outros grupos após a reperfusão por 120 minutos, exceto o grupo controle. Assim, o pré-condicionamento com DEX melhorou significativamente a hemodinâmica após a lesão de I/R do miocárdio, e os efeitos foram revertidos pelo efeito da ioimbina (IO) como antagonista do receptor adrenérgico ( Figura 2 ).

– <italic>A dexmedetomidina melhora a função cardíaca em corações de ratos com isquemia-reperfusão. A-F: efeito da dexmedetomidina na FC, produto de taxa de pressão (PTP = FC + PDVE), pressão ventricular esquerda máxima de mudanças positivas e negativas (±dp/dt<sub>max</sub>) da lesão de I/R em ratos, PDVE, PDFVE. Os dados são apresentados como média ± desvio padrão. n=12. <sup>•</sup>P<0,05, vs. ponto de início da isquemia, <sup>#</sup>P<0,05, vs. grupo controle na reperfusão por 120 minutos. <sup>∆</sup>P<0,05, vs. grupo I/R com reperfusão por 120 minutos. <sup></sup>P<0,05, vs. grupo DEX com reperfusão por 120 minutos. Base: no final do ponto de equilíbrio, 45 minutos: ponto de início da isquemia, 205 minutos: 120 minutos de reperfusão. FC: frequência cardíaca; I/R: isquemia-reperfusão; PDVE: pressão desenvolvida no ventrículo esquerdo; PTP: produto de taxa de pressão; ±dp/dt<sub>max:</sub> pressão ventricular esquerda máxima de mudanças positivas e negativas; PDFVE: pressão diastólica final do ventrículo esquerdo, DEX: dexmedetomidina.</italic>

Pré-tratamento com DEX reduz o tamanho do infarto do miocárdio

O método TTC de coloração, que é padrão ouro para testes de infarto do miocárdio, foi usado para avaliar a função cardioprotetora da DEX. No grupo controle, a porcentagem de infarto foi baixa; porém, aumentou significativamente nos grupos I/R, IO e DEX + IO ( Figura 3A ). Em ratos pré-tratados com DEX, a área do infarto foi significativamente menor em comparação ao grupo I/R. Além disso, não houve diferenças entre os grupos IO e DEX + IO em comparação ao grupo I/R. Assim, a adição de IO reverteu os efeitos protetores da DEX na lesão do miocárdio ( Figura 3 ).

– <italic>Coloração com TTC e HE no método Langendorff após a lesão de isquemia-reperfusão. (A) Imagens representando as amostras coloradas com TTC mostrando a área do infarto (branco) e a área sem infarto (vermelho). (B) Análise do tamanho do infarto do miocárdio no controle e no coração isolado com I/R induzida. Os dados são apresentados como média ± desvio padrão. N = 6<sup>. *</sup>P<0,05, vs. grupo controle; <sup>#</sup>P<0,05, vs. Grupo da I/R; <sup>∆</sup>P<0,05, vs. grupo DEX. (C) Imagens representando as amostras com coloração de HE (ampliação, ×200) demonstrando mudanças histopatológicas no miocárdio. TTC: coloração do cloreto de trifeniltetrazólio. HE: hematoxilina e eosina, I/R: isquemia-reperfusão.</italic>

Pré-tratamento com DEX atenua a lesão no tecido do miocárdio

A lesão miocárdica foi medida por meio da coloração com HE. A coloração com HE revelou que a estrutura do miocárdio estava completa e exibia um arranjo regular, fibras musculares cardíacas normais e nenhuma necrose, com um leve edema no cardiomiócito no grupo controle, enquanto a estrutura do miocárdio foi severamente comprometida depois da lesão de I/R. O grupo I/R mostrou arranjo irregular de miofibrilas e músculos cardíacos rompidos. Como demonstrado na Figura 3C , o pré-condicionamento com DEX melhorou muito essas alterações patológicas; porém, a IO reverteu parcialmente o efeito protetor.

 Pré-tratamento com DEX suprime a apoptose miocárdica

O método de TUNEL foi usado para determinar os efeitos da DEX na apoptose do cardiomiócito em corações isolados de ratos após I/R. Em comparação ao grupo controle (taxa de apoptose, 0,00±0.00%), o número de células TUNEL positivas aumentou significativamente nos grupos de I/R (taxa de apoptose, 58,17±0,60%), IO (taxa de apoptose, 57,11±1,39%) e DEX + IO (taxa de apoptose 57,62±1,50%); ao mesmo tempo, não houve diferenças significativas na taxa de apoptose entre os grupos I/R, IO e DEX + IO ( Figura 4C ). Em comparação aos grupos I/R, IO e DEX + IO, a taxa de apoptose diminuiu muito no grupo DEX. Assim, a DEX pode reduzir a apoptose do miocárdio. Porém, a IO reverteu completamente este efeito ( Figura 4 ).

– <italic>Análise TUNEL para detectar apoptose em corações com isquemia-reperfusão. (A) Imagens representativas das análises TUNEL (ampliação, x400) mostrando o núcleo total (azul) e o núcleo apoptótico (verde). (B) Análise do núcleo apoptótico do cardiomiócito no grupo controle e no coração isolado com I/R induzida. (C) Análise das taxas de apoptose do cardiomiócito no grupo controle e no coração isolado com I/R induzida. Dados são apresentados como média ± desvio padrão. N=6. <sup>*</sup>P<0,05, vs. grupo controle; <sup>#</sup>P<0,05, vs. grupo I/R; <sup>∆</sup>P<0,05, vs. grupo DEX. TUNEL: método de marcação de terminações dUTP e deoxinucleotidil terminal transferase.</italic>

O pré-tratamento com DEX alivia a apoptose ao inibir o estresse do RE via PERK

Para esclarecer o estabelecimento de sucesso de um modelo de estresse do RE induzido pela lesão de I/R do miocárdio, a ativação da GRP78 e da PERK foi avaliada. Neste estudo, a GRP78, importante marcador da ocorrência do estresse do RE, foi altamente regulada no nível da proteína em todos os grupos experimentais em comparação ao grupo controle ( Figura 5 ). Como a GRP78 é alvo direto da PERK, que é um transdutor altamente conservado da apoptose no estresse do RE, os níveis de expressão das proteínas PERK e p-PERK foram examinados. Sob condições fisiológicas, a superexpressão da GRP78 não alterou a ativação da PERK como sendo indiretamente determinada pela forforilação da PERK ( Figura 6 ). A expressão da proteína p-PERK nos grupos I/R, IO e DEX + IO foi maior se comparada àquela no grupo controle e nos grupos DEX ( Figure 6 ). Em contraste, ela foi bloqueada com sucesso pelo tratamento com DEX. Esses resultados sugerem que a DEX pode ter participação na proteção gerada pelo estresse do RE contra a lesão de reperfusão, enquanto o efeito protetivo da DEX foi revertido pela IO, em comparação ao grupo DEX + IO. Para avaliar melhor os efeitos da DEX na via PERK e seu potencial mecanismo molecular, componentes derivados da PERK na resposta de estresse do RE foram investigados, incluindo o fator de iniciação eucariótico 2α (eIF2α), ativando o fator de transcrição 4 (TCF-4) e proteína homóloga à proteína ligadora do acentuador CCAAT (CHOP). Os resultados demonstraram que os níveis de p-eIF2α, TCF-4 e CHOP foram regulados nos grupos I/R, IO e DEX + IO, em comparação ao grupo controle, enquanto o pré-tratamento com a DEX inibiu a expressão das três proteínas ( Figura 5 , Figura 6 ). A análise do modelo TUNEL confirmou esses resultados. Assim, os resultados mostraram que a DEX pode aliviar a lesão de I/R do miocárdio mediada pela apoptose ao suprimir a ativação da via PERK.

– <italic>A dexmedetomidina protegeu o miocárdio da lesão de I/R e reduziu a apoptose. (A) Teste Western blot detectou expressão das proteínas GRP78, TCF-4 e CHOP. (B-D) Análise da expressão de TCF-4, GRP78 e CHOP no controle e no coração isolado com I/R induzida. Os dados são apresentados como média ± desvio padrão. n=6. *P<0,05, vs. grupo controle. #P<0,05, vs. grupo I/R, ∆P<0,05, vs. grupo DEX. I/R: isquemia-reperfusão. GRP78: proteína-78 regulada pela glicose, TCF-4: fator de transcrição 4, CHOP: proteína homóloga à proteína ligadora do acentuador CCAAT.</italic>

– <italic>Efeito da DEX na expressão de p-PERK e p-eIF2α. (A, B) Western-blot detectou a expressão das proteínas PERK, p-PERK, eIF2α, p-eIF2α. (C, D) Análise da expressão de PERK, p-PERK, eIF2α, p-eIF2α no controle e no coração isolado com I/R induzida. Dados são apresentados como ± desvio padrão. n=6. <sup>*</sup>P<0,05, vs. grupo controle. <sup>#</sup>P<0,05, vs. grupo I/R, <sup>∆</sup>P<0,05, vs. grupo DEX. I/R: isquemia-reperfusão. PERK: proteína quinase do retículo endoplasmático, p-PERK: proteína quinase do retículo endoplasmático fosforilada, eIF2a: fator de iniciação eucariótico 2α, p-eIF2α: fator de iniciação eucariótico 2α fosforilado.</italic>

Discussão

Este estudo indicou que a DEX pode proteger contra a lesão de I/R do miocárdio, o que está de acordo com um estudo anterior.21 Os efeitos protetivos da DEX na lesão de I/R do miocárdio por meio da inibição das vias apoptóticas foram confirmados previamente.22 Da mesma forma, os resultados deste estudo indicaram que a DEX melhorou a hemodinâmica cardíaca e reduziu a apoptose, o que foi revelado pelas mudanças na morfologia apoptótica e nas taxas de apoptose do método TUNEL. O estresse excessivo do RE demonstrou ter um papel importante na lesão de I/R do miocárdio, levando à apoptose.23 GRP78, que é uma proteína acompanhante do RE, pertence à família das proteínas do choque térmico (Hsp70), o que indica a ocorrência do estresse do RE e regula a homeostase do RE em certo ponto.24 Estudos anteriores demonstraram que a I/R induz a expressão da proteína GRP78.25 , 26 Os resultados deste estudo demonstraram que a proteína GRP78 foi induzida pela I/R, enquanto o tratamento com DEX reduziu a expressão de GRP78, o que indicou que a DEX pode exercer seus efeitos protetores ao suprimir o estresse do RE.

A PERK é um receptor de estresse com atividade de serina-treonina quinase, que é ativada por trans-autofosforilação e oligomerização. Em condições prolongadas de estresse do RE, a via PERK/eIF2α/TCF-4/CHOP contribui com a apoptose durante a lesão de I/R do miocárdio.27 , 28 Um estudo prévio reportou que a GRP78 localizada na membrana é essencial para a fosforilação da PERK,26 o que também foi observado neste estudo. Observa-se que a maior expressão da proteína GRP78 foi detectada no miocárdio do coração de rato isolado e perfundido com isquemia por 40 minutos e reperfusão por 120 minutos.29 Além disso, descobrimos que a isquemia por 40 minutos foi ótima para os corações com isquemia-reperfusão no método Langendorff;30 então, selecionamos isquemia por 40 minutos e reperfusão de 120 minutos no modelo atual ex vivo da lesão do estresse do retículo endoplasmático (ERE). Também foi reportado que a ativação do domínio PERK quinase nos primeiros estágios do estresse do RE leva à fosforilação da eIF2α, o que reduz a iniciação da transcrição e o enovelamento de proteínas, mantendo a homeostase no retículo endoplasmático. A fosforilação avançada da eIF2α por meio da ativação da TCF-4 induz a superexpressão da proteína apoptótica CHOP, levando à apoptose.31 , 32 Estudos relacionados mostraram que os mecanismos da DEX contra a lesão de I/R do miocárdio são mediados pela ativação da α2-Ars e dos processos anti-inflamatórios,22 enquanto o efeito da DEX no estresse do RE na lesão de I/R do miocárdio foi raramente investigado. Os resultados deste estudo demonstraram que a DEX inibiu o estresse do RE mediado pelas vias PERK/eIF2α/TCF-4/CHOP durante a lesão de I/R do miocárdio. Um estudo anterior descreveu que a I/R do miocárdio causa um acúmulo anormal de proteínas não enoveladas no lúmen do retículo endoplasmático, e contribui para a autofosforilação da PERK.33 Neste estudo, o nível de expressão da proteína p-PERK aumentou no grupo I/R, mas diminuiu com o pré-tratamento com a DEX. Com a ativação da PERK, a fosforilação da proteína derivada eIF2α inibiu o agrupamento de ribossomos 80S e a síntese proteica em massa.34 , 35 Outro estudo indicou que a desfosforilação da eIF2α suprimiu a sinalização dos derivados da TCF-4/CHOP.36 Assim, a eIF2α pode ser um componente chave na sinalização da PERK. Os resultados deste estudo demonstraram que depois do tratamento com DEX, o nível de p-eIF2α diminui em relação ao grupo I/R. A TCF-4 tem papel essencial na promoção da apoptose, que é induzida pela fosforilação da eIF2α.37 - 39 Os resultados deste estudo sugerem que a TCF-4 pode ser responsável pela apoptose do cardiomiócito durante o estresse do RE induzido pela I/R, enquanto o tratamento com a DEX pode diminuir a TCF-4. CHOP, que é membro da família de fatores de transcrição da proteína ligadora do acentuador CCAAT,40 sendo um marcador clássico da iniciação da apoptose, ou seja, tem papel essencial na apoptose do miocárdio mediada pelo estresse do RE.41 , 42 Nossos resultados sugerem que a expressão da proteína CHOP aumentou no grupo I/R, o que corresponde aos resultados da análise da apoptose. Este estudo demonstrou que a DEX tinha um efeito anti-apoptótico, e o mecanismo pode depender do estresse do RE ativado pela via PERK.

A IO é um bloqueador seletivo do α2-AR pré-sináptico, que dilata o músculo liso vascular, reduz o tom simpático e aumenta o tom parassimpático periférico. Neste estudo, a IO reverteu os efeitos protetores do pré-tratamento com a DEX no miocárdio de ratos, o que resultou em maior tamanho de infarto do miocárdio e apoptose do cardiomiócito; além disso, os níveis de expressão das proteínas PERK aumentaram. Juntos, esses resultados indicam que o efeito protetor do pré-condicionamento com a DEX em miocárdios de ratos pode ser antagonizado por um bloqueador RA, o que está de acordo com um estudo prévio.43

Embora os resultados deste estudo identifiquem um papel importante da via PERK na sobrevivência de cardiomiócitos, houve algumas limitações. Mais trabalhos são necessários para avaliar outros potenciais mecanismos além da apoptose na via PERK. Além disso, a apoptose deve ocorrer no contexto de excessivo estresse do RE. Ao contrário, o RE promove a sobrevivência celular normal ao oferecer nutrientes de organelas e proteínas digestivas danificadas e quando os níveis de estresse do RE estão baixos, chamado autofagia do RE induzida pelo estresse. Outras avaliações são necessárias para determinar o limite entre a autoadaptação protetora da PERK e a apoptose prejudicial na lesão de I/R do miocárdio.

 Conclusão

Este estudo confirmou que o pré-condicionamento com a DEX reduziu a lesão de I/R do miocárdio e melhorou a função cardíaca ao inibir a via apoptótica ativada pela PERK.

 Agradecimentos

Este estudo foi apoiado pelo Plano de Desenvolvimento da Ciência e Tecnologia da Província de Guizhou (n. 2016[1175]), e o Fundo de pesquisa da Pós-Graduação da Província de Ghizou (n. KYJJ2017034).

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Vinculação acadêmica

Não há vinculação deste estudo a programas de pós-graduação.

Aprovação ética e consentimento informado

Este estudo foi aprovado pelo Comitê de Ética do Experimental Animal Care and Use committee of Zunyi Medical University sob o número de protocolo 1492, 2001. Todos os procedimentos envolvidos nesse estudo estão de acordo com a Declaração de Helsinki de 1975, atualizada em 2013.

Fontes de financiamento: O presente estudo foi financiado por The Science and Technology Development Plan of Guizhou Province (grant no.2016[1175]), and The Postgraduate Research Fund of Guizhou Province (grant no. KYJJ2017034).

 Original Article Dexmedetomidine Preconditioning Reduces Myocardial Ischemia-Reperfusion Injury in Rats by Inhibiting the PERK Pathway Chen YuJiao 1 2 Cao Song 1 0000-0002-0707-7671 Chen Hui 1 Yin CunZhi 1 Xu XinPeng 1 Yang ZaiQun 1 China Zunyi Medical University, Zunyi, Guizhou – China China Affiliated Hospital of North Sichuan Medical College, NanChong, SiChuan - China Mailing Address: Hui Chen • Zunyi Medical College Affiliated Hospital - 149 Dalian road, Zunyi, Guizhou Zunyi 563003 – China E-mail: 2267390140@qq.com

Author Contributions

Conception and design of the research: Chen Y, Chen H; Acquisition of data: Chen Y, Cao S, Chen H, Yin C, Xu X, Yang Z; Analysis and interpretation of the data: Chen Y, Yang Z; Statistical analysis: Chen Y, Cao S, Yin C; Obtaining financing and Writing of the manuscript: Chen Y; Critical revision of the manuscript for intellectual content: Chen Y, Xu X.

Potential Conflict of Interest

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

 Abstract Background

Ischemic heart disease has attracted much attention due to its high mortality rates, treatment costs and the increasing morbidity in the young population. Strategies for reperfusion have reduced mortality. However, reperfusion can lead to cardiomyocyte death and subsequent irreversible myocardial damage. At present, the timely and targeted treatment of ischemia-reperfusion (I/R) injury is often lacking.

 Objectives

To evaluate if dexmedetomidine (DEX) has a protective effect in myocardiual I/R and explore the possible mechanism behind it.

 Methods

Rat hearts were perfused with a Langendorff perfusion system, and randomly assigned to five groups: control group, perfused with Krebs-Henseleit (K-H) solution for 205 minutes without ischemia; and four test groups that underwent 40 minutes of global ischemia and 120 min of reperfusion. The DEX group, the yohimbine (YOH) group and the DEX + YOH group were perfused with DEX (10 nM), YOH (1 μM) or the combination of DEX and YOH prior to reperfusion, respectively. Cardiac hemodynamics, myocardial infarct size, and myocardial histology were evaluated. The expression of glucose-related protein 78 (GRP78), protein kinase R-like ER kinase (PERK), phosphorylated PERK, eukaryotic initiation factor 2α (eIF2α), phosphorylated eIF2α, activating transcription factor 4 (ATF4), and CCAAT/enhancer-binding protein homologous protein (CHOP) were assessed. P<0.05 was considered to indicate a statistically significant difference.

 Results

DEX preconditioning improved the cardiac function of I/R hearts, reduced myocardial infarction, myocardial apoptosis, and the expression of GRP78, p-PERK, eIF2α, p-eIF2α, ATF4 and CHOP.

 Conclusions

DEX pretreatment reduced myocardial I/R injury by suppressing apoptosis, which was induced by the PERK pathway.

 Dexmedetomidine Ischemic Cardiomyopathy/mortality Myocardial Reperfusion Hipnotics and Sedatives Protein Kinases Rats Introduction

Ischemic heart disease has attracted much attention due to its high mortality rates, treatment costs and the increasing morbidity in the young population. In the past three decades, reperfusion strategies to alleviate ischemic injury have been widely included in clinical practice. However, reperfusion can lead to cardiomyocyte death, and subsequent irreversible myocardial damage.1

Myocardial ischemia-reperfusion injury (MIRI) is a long-term and complex pathophysiological process. The process of restoring blood flow to the ischemic myocardium can induce a wide range of basic biological changes, including impaired ion homeostasis, oxygen radical bursts, autophagy, ATP metabolic disorders, inflammation, oxidative stress, mitochondrial dysfunction, and apoptosis.2 Recent studies have demonstrated that the damage inflicted on the myocardium involves two processes: ischemia and reperfusion injury (I/R injury). I/R injury is a major determinant of long-term mortality; thus, the possibility of ameliorating the extent of the injury is of great individual and socioeconomic value.3 - 7

It has been demonstrated that cardiomyocyte apoptosis is a major mechanism of myocardial I/R-injury.8 Cardiomyocyte apoptosis is triggered by the mitochondrial and death receptor pathways under the conditions of myocardial reperfusion.9 However, these mechanisms have yet to be fully determined.2 Recent studies have explored the association between the endoplasmic reticulum (ER) and myocardial I/R-injury.9 - 11 Stress conditions such as I/R, hypoxia, and oxidative stress have been identified in the dysregulation of ER functions, thus triggering ER stress.12 The pathological consequences of ER lumen disruption and miscommunication have been implicated in myocardial I/R-injury.13 , 14 However, it is still unclear whether or not ER stress induces apoptosis by activating PERK during myocardial reperfusion injury.15

At present, due to the shortage of specific drugs and standard therapies, the timely and targeted treatment of I/R injury is often lacking. Dexmedetomidine (DEX) is a highly selective α2adrenergic receptor (AR) agonist, which has limited impact on hemodynamics and breathing, thus providing the ideal sedation and analgesia for patients undergoing cardiovascular surgery.16 Previous studies have demonstrated that DEX preconditioning showed cardioprotective effects in ischemic hearts.17 - 20 However, the molecular mechanism of DEX protection, to date, remains unknown. The present study hypothesized that pre-treatment with DEX protects the heart from I/R injury by PERK pathway activation, which is dependent on the activation of the α2receptor. Therefore, the present study used an isolated heart I/R model to evaluate the effects of DEX on I/R injury and its potential mechanism.

 Methods Experimental animals

Male adult Sprague-Dawley rats (8-10 weeks old; 260-280g) were obtained from the animal experiment center of Changsha (Changsha, China). All experimental procedures were in accordance with the “Guide for the Care and Use of Laboratory Animals”, published in China (No.1492, 2001), and were approved by the Experimental Animal Care and Use committee of the Zunyi Medical University. All rats were housed under standard conditions (room temperature, 22℃; 12 h light/dark cycle) with free access to food and water in the Guizhou Key Laboratory of Anesthesiology and Organ Protection of Zunyi Medical University.

 Isolation of adult rat hearts

Rats were anesthetized with sodium pentobarbital (45 mg/kg, intraperitoneally). After the successful anesthesia, the heart was immediately excised from the chest by sternotomy and immersed in ice-cold Krebs-Henseleit (K-H) solution (NaCl, 119 mM; KCl, 6.0 mM; CaCl2, 1.24 mM; NaHCO3, 20.1 mM; KH2PO4, 1.24 mM; MgSO4, 1.24 mM; glucose, 11.2 mM). The excised heart was retrogradely placed in a Langendorff perfusion setup via the aorta (PanLab). The K-H solution was perfused at a constant perfusion pressure of 75-80 mmHg and balanced with a mixture of 95% O2and 5% CO2to a pH of 7.35-7.45 at 37°C. A latex balloon connected to a pressure transducer was inserted in the left ventricle through the mitral valve, and filled with normal saline to produce a left ventricular end-diastolic pressure (LVEDP) of 2-10 mmHg. The balloon volume was maintained constant throughout the experiment. Heart rate (HR), LVEDP, left ventricular developed pressure (LVDP = LVSP - LVEDP), left ventricular pressure peak rates of positive and negative changes (±dp/dtmax) and rate pressure product (RPP = HR x LVDP) were recorded by the Lab Chart system.

 I/R and DEX preconditioning in isolated rat heart

For each test, following a 15-minute equilibration of perfusion, the hearts in which the baseline LVDP and HR were >50mmHg and >200 beats/min were randomly assigned to five groups in a sealed envelope randomization assignment. The specimens in all groups were perfused for 30 minutes prior to 40 minutes of normothermic global ischemia, followed by 120 minutes of reperfusion with the exception of the control group (n= 6): (i) control group, no preconditioning protocol, perfused with normal K-H solution; (ii) I/R group, no preconditioning protocol, perfused with normal K-H solution prior to ischemia; (iii) DEX group, preconditioned with 10 nM DEX prior to ischemia; (iv) yohimbine (YOH) group, preconditioned with1 μM YOH prior to ischemia; and(v) DEX + YOH group, preconditioned with 10 nM DEX + 1μM YOH prior to ischemia. The concentrations of DEX (Hengrui pharmaceutical) and antagonist were selected based on previous studies. Yohimbine (YOH) was used as an α2-AR antagonist. DEX and YOH were dissolved in the modified Krebs-Ringer solution ( Figure 1 ).

– <italic>Langendorff myocardial ischemia-reperfusion injury model. For each test, after a 15-minute equilibration of perfusion, the prepped hearts were randomly assigned to five groups for baseline LVDP and HR, which were less than 50 mmHg and 200 beats/min. The specimens in control group were continuously perfused with K-H solution for 205 minutes. The specimens in four other groups were perfused for 30 minutes prior to 40 minutes of normothermic global ischemia, followed by 120 minutes of reperfusion. The specimens in the I/R group had no preconditioning protocol perfused with normal K-H solution before ischemia; the specimens in DEX group were perfused with DEX (10 nM) before ischemia; the specimens in YOH group were perfused with YOH (1 μM) before ischemia; the specimens in DEX + YOH group were perfused with DEX (10 nM)+YOH (1 μM) before ischemia. LVDP: left ventricular developed pressure, HR: heart rate, K-H: Krebs-Henseleit, I/R: ischemia-reperfusion, DEX: dexmedetomidine, YOH: yohimbine</italic>

Infarct size determination

Infarct size was assessed by the triphenyl tetrazolium chloride (TTC) staining method. After reperfusion, the tissues that were adherent to the arterial root were removed, and the whole heart was frozen. The frozen heart was cut into 1-2 mm thick slices. The slices were immersed in a vial with 1% TTC and incubated in a water bath for 30 minutes at 37°C. The vial was continuously agitated to obtain an even staining. The slices were subsequently placed in 10% formaldehyde for 24 hours, placed on a glass plate, and a cover glass was placed over the slices. Shims of 2 mm were placed in the corners between the glasses to obtain the desired slice thickness, and digital photographs were taken. Areas of infarction and normal areas were determined in a blinded manner by planimetry with the Image-Pro Plus 6.0 software (Media Cybernetics, Inc.), and infarct size was expressed as a percentage of the total area.

 Histopathological examination

Myocardial tissues harvested from the rats were cut into 1 cm sections, fixed in 4% paraformaldehyde and embedded in paraffin. To quantify the extent of myocardial damage, the biopsies were cut into 5 µm sections, which were stained with hematoxylin and eosin (H&E) for 90 minutes, at room temperature, and six fields of view (magnification, x200) were randomly selected for evaluation in each group.

 TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed to determine the extent of apoptosis in the myocardium using a commercially available kit (Baiao Cisco Biological Technology) according to the manufacturer’s instructions. Green nuclear labeling was defined as TUNEL-positive cells. To determine the extent of myocardial apoptosis, five fields (magnification, x 200) were randomly selected from two sections in each group, and the apoptosis index (AI) was calculated using Image-Pro Plus 6.0 (AI) as follows: AI = number of apoptotic cells / total number of cells counted.

 Western blotting

The heart was homogenized and lysed with a RIPA buffer (Thermo Fisher scientific, Inc.). The tissues were centrifuged at 125 x g for six minutes and the supernatant was collected. The concentration of the protein was determined by a BCA protein assay (Thermo Fisher scientific, Inc.). The samples were separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked with 5% skimmed milk for two hours, membranes were probed with anti-GRP78 (1:1000; cat no. PA1-014A; Thermo Fisher scientific), anti-PERK (1:1000; cat no. PA5-79193; Thermo Fisher scientific), anti-p-PERK (1:1000; cat no. MA5-15033; Thermo Fisher scientific), anti-eIF2α (1:1000; cat no. MA1-079; Thermo Fisher scientific), anti-p-eIF2α (1:1000; cat no. 44-728G; Thermo Fisher scientific), anti-ATF4 (1:1000; cat no. PA5-68802; Thermo Fisher scientific), anti-CHOP (1:1000; cat no. PA5-86145; Thermo Fisher scientific), anti-GAPDH (1:1000; cat no. MA5-32539; Thermo Fisher scientific) primary antibodies at 4°C overnight. After washing the membranes three times, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000; cat no. G-21234; Thermo Fisher scientific) for two hours. The content of the protein was determined by a gel imaging system (Tanon Science & Technology).

 Statistical analysis

The sample size of this study was based on the preliminary experiment. Means of LVEDP in the control group, the DEX group and the I/R group following reperfusion for 120 minutes were 12.52, 16.23, 22.39, respectively, and Standard deviations were 1.45, 2.44 and 145, respectively. The α-level test was considered as 0.05, Z0.05/2 = 1.96. The power level, 1 − β, was considered as 0.8. For the control and the EDX groups, a sample size of six was required for each group. For the I/R and the DEX groups, a sample size of four was required for each group. So, a sample size of six was determined per group.

Data are presented as mean ± standard deviation (SD). SPSS 20.0 (IBM, Corp) was used for statistical analyses. Differences between groups were analyzed with the one-way analysis of variance (ANOVA). The Shapiro-Wilk test was employed to verify normality. The LSD test was used for the comparison of homogeneous variance, and the Dunnett’s T3 test was used for the comparison of uneven variance. P< 0.05 was considered to indicate a statistically significant difference.

 Results DEX improves cardiac recovery from the I/R injury

To determine the effects of DEX on myocardial I/R-injury, an isolated perfused heart preparation was performed by perfusing rat hearts for 30 minutes with modified K-H solution prior to 40 minutes of global ischemia, followed by 120 minutes of reperfusion. The analysis of variance demonstrated there were no significant differences in hemodynamics between the groups at the end of the balance point. After 45 minutes of perfusion, no significant differences were observed in HR, ±dp/dtmax, LVDP, LVEDP and RPP, with the exception of the YOH group and the DEX + YOH group. In the control group, HR, ±dp/dtmax, LVDP, and RPP were higher and LVEDP was lower compared with the other groups after reperfusion for 120 minutes, which suggested that 40-minute ischemia at room temperature was challenging for rat hearts. During the second half of the 120-minute reperfusion, a number of hearts exhibited failure and arrhythmia (~10%). However, in the DEX group, HR, ±dp/dtmax, LVDP, and RPP were higher and LVEDP was lower compared with the other groups after reperfusion for 120 minutes, with the exception of the control group. Therefore, DEX preconditioning significantly improved the hemodynamics following myocardial I/R injury, and the effects were reversed by the AR antagonist YOH ( Figure 2 ).

– <italic>Dexmedetomidine improves cardiac function of ischemia-reperfusion rat hearts. A-F: effect of dexmedetomidine on the HR, rate pressure product (RPP = HR×LVDP), left ventricular pressure peak rates of positive and negative changes (±dp/dt<sub>max</sub>) of I/R injury in rats, LVDP, LVEDP. Data are presented as the mean ± standard deviation. n=12.<sup>•</sup>P<0.05, vs. ischemia beginning point,<sup>#</sup>P<0.05, vs. control group at reperfusion for 120 minutes.<sup>∆</sup>P<0.05, vs. I/R group at reperfusion for 120 minutes.<sup></sup>P<0.05, vs. DEX group at reperfusion for 120 minutes. Baseline: at the end of the balance point, 45 minutes: ischemia beginning point, 205 minutes: 120 minutes of reperfusion. HR, heart rate, I/R: ischemia-reperfusion, LVDP: left ventricular developed pressure, RPP: rate pressure product, ±dp/dt<sub>max</sub>: left ventricular pressure peak rates of positive and negative changes, LVDP: left ventricular developed pressure, LVEDP: left ventricular end diastolic pressure, DEX: dexmedetomidine.</italic>

Pre-treatment with DEX reduces myocardial infarct size

TTC staining, which is the gold standard for testing myocardial infarct, was used to evaluate the cardio-protective function of DEX. In the control group, the percentage of infarct was low; however, it was significantly increased in the I/R, YOH and DEX + YOH groups ( Figure 3A ). In rats that were pre-treated with DEX, the infarct area was significantly smaller when compared with that in the I/R group. Additionally, no differences were observed between the YOH and the DEX + YOH groups compared with the I/R group. Therefore, the addition of YOH reversed the DEX-mediated protective effects on myocardial injury ( Figure 3 ).

– <italic>TTC staining and HE staining of Langendorff hearts after ischemia-reperfusion injury. (A) Representative images of TTC stained samples showing the infarct area (white) and the non-infarct area (red). (B) Analysis of the myocardial infarct size in control and in I/R-induced isolated heart. Data are presented as the mean ± standard deviation. n=6.<sup>*</sup>P<0.05, vs. control group;<sup>#</sup>P<0.05, vs. I/R group;<sup>∆</sup>P<0.05, vs. DEX group. (C) Representative images of HE stained samples (magnification, ×200) demonstrating histopathological changes in the myocardium. TTC: triphenyl tetrazolium chloride staining technique. HE: hematoxylin and eosin, I/R: ischemia-reperfusion.</italic>

Pretreatment with DEX attenuates myocardial tissue damage

Myocardial injury was further measured in the H&E-stained sections. H&E staining revealed that myocardial structure was complete and exhibited regular arrangement, normal cardiac muscle fibers and no necrosis, with mild cardiomyocyte edema in the control group, whereas the structure of the myocardium was severely damaged after the I/R injury. The I/R group exhibited disordered myofibril arrangement and ruptured cardiac muscle fibers. As shown in Figure 3C , DEX preconditioning significantly improved these pathological changes; however, YOH partially reversed the protective effect.

 Pretreatment with DEX suppresses myocardial apoptosis

The TUNEL assay was used to determine the effects of DEX on cardiomyocytic apoptosis in isolated rat hearts after I/R. Compared with the control group (apoptotic rate, 0.00±0.00%), the number of TUNEL-positive cells increased significantly in the I/R (apoptotic rate, 58.17±0.60%), YOH (apoptotic rate, 57.11±1.39%) and DEX + YOH (apoptotic rate, 57.62±1.50%) groups, whereas no significant differences were observed in apoptotic rate among the I/R, YOH and the DEX + YOH groups ( Figure 4C ). Compared with the I/R, YOH and DEX + YOH groups, apoptotic rate was significantly decreased in the DEX group. Therefore, DEX may decrease myocardial apoptosis. However, YOH completely reversed this effect ( Figure 4 ).

– <italic>TUNEL assays to detect apoptosis in ischemia-reperfusion hearts. (A) Representative images of TUNEL assays (magnification, ×400) showing the total nucleus (blue) and apoptotic nucleus (green). (B) Analysis of the cardiomyocytic apoptotic nucleus in the control group and in I/R-induced isolated heart. (C) Analysis of the cardiomyocytic apoptosis rates in control group and in I/R-induced isolated heart. Data are presented as the mean ± standard deviation. n=6.<sup>*</sup>P<0.05, vs. control group;<sup>#</sup>P<0.05, vs. I/R group;<sup>∆</sup>P<0.05, vs. DEX group. TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.</italic>

DEX pret-reatment alleviates apoptosis by inhibiting the ER stress-mediated PERK pathway

To elucidate the successful establishment of an ER stress model induced by myocardial I/R injury, the activation of GRP78 and PERK was evaluated. In the present study, GRP78, which is an important marker for the occurrence of ER stress, was extensively upregulated at the protein level in all experimental groups compared with the control group ( Figure 5 ). As GRP78 is a direct target of PERK, which is a highly conserved ER stress-mediated apoptosis transducer, the expression levels of PERK and p-PERK protein were examined. Under physiological conditions, the overexpression of GRP78 did not change the activation of PERK as indirectly determined by PERK phosphorylation ( Figure 6 ). The expression of p-PERK protein in the I/R, YOH and DEX + YOH groups was higher compared with that in the control and the DEX groups ( Figure 6 ). By contrast, this was successfully blocked by the DEX treatment. These results suggested that DEX may participate in ER stress-mediated protection against reperfusion injury, whereas the protective effect of DEX was reversed by YOH compared with the DEX + YOH group. To further determine the effects of DEX on the PERK pathway and its potential molecular mechanism, downstream components of the PERK pathway in ER stress response were investigated, including the eukaryotic initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4) and CCAAT/enhancer-binding protein homologous protein (CHOP). The results demonstrated that the levels of p-eIF2α, ATF4 and CHOP were notably upregulated in the I/R, YOH and DEX + YOH groups in comparison with the control group, whereas pre-treatment with DEX inhibited the expression of the three proteins ( Figure 5 , Figure 6 ). The examination by TUNEL assay confirmed these results. Therefore, the results indicated that DEX may relieve myocardial I/R injury-mediated apoptosis by suppressing the activation of the PERK pathway.

– <italic>Dexmedetomidine protected the myocardium from I/R injury and reduced apoptosis. (A) Western-blotting detecting of GRP78, ATF4, CHOP protein expression. (B-D) Analysis of the expression of ATF4, GRP78 CHOP in control and I/R-induced isolated heart. Data are presented as the mean ± standard deviation. n=6.<sup>*</sup>P<0.05, vs. control group.<sup>#</sup>P<0.05, vs. I/R group,<sup>∆</sup>P<0.05, vs. DEX group. I/R: ischemia-reperfusion. GRP78: glucose-related protein 78, ATF4: activating transcription factor 4, CHOP: CCAAT/enhancer-binding protein homologous protein.</italic>

– <italic>Effect of DEX on the expression of p-PERK and p-eIF2α. (A, B) Western-blotting detecting of PERK, p-PERK, eIF2α, p-eIF2α protein expression. (C, D) Analysis of the expression of PERK, p-PERK, eIF2α, p-eIF2α in control and I/R-induced isolated heart. Data are presented as the mean ± standard deviation. n=6.<sup>*</sup>P<0.05, vs. control group.<sup>#</sup>P<0.05, vs. I/R group,<sup>∆</sup>P<0.05, vs. DEX group. I/R: ischemia-reperfusion. PERK: protein kinase R-like ER kinase, p-PERK: phosphorylation-protein kinase R-like ER kinase, eIF2a: eukaryotic initiation factor 2a, p-eIF2α: phosphorylation-eukaryotic initiation factor 2a.</italic>

Discussion

The present study indicated that DEX may protect against myocardial I/R injury, which was in accordance with a previous study.21 The protective effects of DEX on myocardial I/R injury through the inhibition of apoptotic pathways have been previously confirmed.22 Similarly, the results of the present study indicated that DEX improved cardiac hemodynamics and decreased apoptosis, which was revealed by the changes in apoptotic morphology and apoptotic rates in the TUNEL assay. Excessive ER stress has been demonstrated to serve an important role in myocardial I/R injury and lead to apoptosis.23 GRP78, which is an ER chaperone protein, belongs to one of the heat-shock protein 70 (Hsp70) family, which indicates the occurrence of ER stress and regulates ER homeostasis at a certain point.24 Previous studies have demonstrated that I/R induces the expression of GRP78 protein.25 , 26 The results of the present study demonstrated that the GRP78 protein was induced by I/R, whereas the DEX treatment decreased GRP78 expression, which indicated that DEX may exert its protective effects by suppressing ER stress.

PERK is a stress receptor on the ER with serine-threonine kinase activity, which is activated by trans-autophosphorylation and oligomerization. Under prolonged ER stress conditions, the PERK/eIF2α/ATF4/CHOP pathway contributes with apoptosis during myocardial I/R injury.27 , 28 A previous study has reported that membrane-localized GRP78 is crucial for PERK phosphorylation,26 which was also observed in the present study. It is reported that the highest expression of GRP78 protein was detected in the myocardium from the isolated and perfused rat heart with 40-minute ischemia and 120-minute reperfusion.29 In addition, we previously found that 40-minute ischemia was optimal for the Langendorff ischemia-reperfusion rat hearts,30 therefore, we selected 40-minute ischemia and 120-minute reperfusion in the present ex vivo ERS ischemia reperfusion injury model. It is also reported that the activation of the PERK kinase domain in the early stages of ER stress leads to the phosphorylation of eIF2α, which reduces transcriptional initiation and protein folding, and maintains homeostasis in the endoplasmic reticulum. Advanced phosphorylation of eIF2α through the activation of ATF4 induces the overexpression of the apoptosis protein CHOP, which leads to apoptosis.31 , 32 Related studies have shown that the mechanisms of DEX against myocardial I/R injury are mediated by the activation of α2-ARs and anti-inflammatory processes,22 whereas the effect of DEX on ER stress in myocardial I/R injury has rarely been investigated. The results of the present study have demonstrated that DEX inhibited the ER stress-mediated PERK/eIF2α/ATF4/CHOP pathway during myocardial I/R injury. A previous study has described that myocardial I/R causes abnormal accumulation of unfolded proteins in the lumen of the endoplasmic reticulum, and contributes with the autophosphorylation of PERK.33 In the present study, p-PERK protein expression level was increased in the I/R group, but decreased with the DEX pre-treatment. With the activation of PERK, the phosphorylation of downstream protein eIF2α inhibited 80S ribosome assembly and massive protein synthesis.34 , 35 Another study has indicated that the dephosphorylation of eIF2α suppressed downstream ATF4/CHOP signaling.36 Therefore, eIF2α may be a key component in PERK-mediated signaling. The results of the present study demonstrated that after the DEX treatment, the level of p-eIF2α was reduced compared with the I/R group. ATF4 plays a role in promoting apoptosis, which is induced by the phosphorylation of eIF2α.37 - 39 The results of the present study suggested that ATF4 may be responsible for cardiomyocyte apoptosis during I/R induced ER stress, whereas DEX treatment may downregulate ATF4. CHOP, which is a member of the CCAAT/enhancer binding protein family of transcription factors,40 is a classic marker of apoptosis initiation, which plays an important role in ER stress-mediated myocardial apoptosis.41 , 42 Our results suggested that the expression of CHOP protein increased in the I/R group, which corresponded to the results of the apoptosis assay. The present study demonstrated that DEX had an anti-apoptotic effect, and the mechanism may be dependent on the ER stress-mediated PERK pathway.

YOH is a selective blocker of presynaptic α2-AR, which dilates vascular smooth muscle, decreases sympathetic tone, and increases peripheral parasympathetic tone. In the present study, YOH reversed the protective effects of DEX pretreatment in the myocardium of rats, which resulted in increased myocardial infarct size and cardiomyocyte apoptosis; in addition, the expression levels of the PERK pathway proteins were increased. Together, these results indicated that the protective effect of DEX preconditioning in rat myocardium may be antagonized by an AR blocker, which is consistent with a previous study.43

Although the results of the present study identified an important role for the PERK pathway in the survival of cardiomyocytes, there were certain limitations to the study. Future work is necessary to analyze other potential mechanisms besides apoptosis and the PERK pathway. In addition, apoptosis is considered to occur in the context of excessive stress in the ER; by contrast, ER promotes normal cell survival by providing nutrients from digesting damaged organelles and proteins when ER stress levels are mild, which is termed ER stress-induced autophagy. Further analysis is required to determine the threshold between PERK exerting protective self-adaptation and injurious apoptosis in myocardial I/R injury.

 Conclusion

This study confirmed that DEX preconditioning reduced myocardial I/R injury and improved cardiac function by inhibiting PERK-mediated apoptosis pathway.

 Acknowledgements

This study was supported by The Science and Technology Development Plan of Guizhou Province (grant no.2016[1175]), and The Postgraduate Research Fund of Guizhou Province (grant no. KYJJ2017034).

Study Association

This study is not associated with any thesis or dissertation work.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Experimental Animal Care and Use committee of Zunyi Medical University under the protocol number 1492, 2001. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013.

Sources of Funding: This study was partially funded by The Science and Technology Development Plan of Guizhou Province (grant no.2016[1175]), and The Postgraduate Research Fund of Guizhou Province (grant no. KYJJ2017034).