Dexmedetomidine Preconditioning Reduces Myocardial Ischemia-Reperfusion Injury in Rats by Inhibiting the PERK Pathway

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.

Figura 1. – 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

Figura 2. – 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_max) da lesão de I/R em ratos, PDVE, PDFVE. Os dados são apresentados como média ± desvio padrão. n=12. ^•P<0,05, vs. ponto de início da isquemia, ^#P<0,05, vs. grupo controle na reperfusão por 120 minutos. ^∆P<0,05, vs. grupo I/R com reperfusão por 120 minutos. ^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_max: pressão ventricular esquerda máxima de mudanças positivas e negativas; PDFVE: pressão diastólica final do ventrículo esquerdo, DEX: dexmedetomidina.

Figura 3. – 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^{. *}P<0,05, vs. grupo controle; ^#P<0,05, vs. Grupo da I/R; ^∆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.

Figura 4. – 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. ^*P<0,05, vs. grupo controle; ^#P<0,05, vs. grupo I/R; ^∆P<0,05, vs. grupo DEX. TUNEL: método de marcação de terminações dUTP e deoxinucleotidil terminal transferase.

Figura 5. – 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.

Figura 6. – 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. ^*P<0,05, vs. grupo controle. ^#P<0,05, vs. grupo I/R, ^∆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.

Figure 1. – 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

Figure 2. – 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_max) of I/R injury in rats, LVDP, LVEDP. Data are presented as the mean ± standard deviation. n=12.^•P<0.05, vs. ischemia beginning point,^#P<0.05, vs. control group at reperfusion for 120 minutes.^∆P<0.05, vs. I/R group at reperfusion for 120 minutes.^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_max: left ventricular pressure peak rates of positive and negative changes, LVDP: left ventricular developed pressure, LVEDP: left ventricular end diastolic pressure, DEX: dexmedetomidine.

Figure 3. – 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.^*P<0.05, vs. control group;^#P<0.05, vs. I/R group;^∆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.

Figure 4. – 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.^*P<0.05, vs. control group;^#P<0.05, vs. I/R group;^∆P<0.05, vs. DEX group. TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 5. – 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.^*P<0.05, vs. control group.^#P<0.05, vs. I/R group,^∆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.

Figure 6. – 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.^*P<0.05, vs. control group.^#P<0.05, vs. I/R group,^∆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.