Role of Opioid Receptors Signaling in Remote Electrostimulation - Induced Protection against Ischemia/Reperfusion Injury in Rat Hearts

<div><p>Aims</p><p>Our previous studies demonstrated that remote electro-stimulation (RES) increased myocardial GSK3 phosphorylation and attenuated ischemia/ reperfusion (I/R) injury in rat hearts. However, the role of various opioid receptors (OR) subtypes in preconditioned RES-induced myocardial protection remains unknown. We investigated the role of OR subtype signaling in RES-induced cardioprotection against I/R injury of the rat heart.</p><p>Methods & Results</p><p>Male Spraque-Dawley rats were used. RES was performed on median nerves area with/without pretreatment with various receptors antagonists such as opioid receptor (OR) subtype receptors (KOR, DOR, and MOR). The expressions of Akt, GSK3, and PKCε expression were analyzed by Western blotting. When RES was preconditioned before the I/R model, the rat's hemodynamic index, infarction size, mortality and serum CK-MB were evaluated. Our results showed that Akt, GSK3 and PKCε expression levels were significantly increased in the RES group compared to the sham group, which were blocked by pretreatment with specific antagonists targeting KOR and DOR, but not MOR subtype. Using the I/R model, the duration of arrhythmia and infarct size were both significantly attenuated in RES group. The mortality rates of the sham RES group, the RES group, RES group + KOR antagonist, RES group + DOR/MOR antagonists (KOR left), RES group + DOR antagonist, and RES group + KOR/MOR antagonists (DOR left) were 50%, 20%, 67%, 13%, 50% and 55%, respectively.</p><p>Conclusion</p><p>The mechanism of RES-induced myocardial protection against I/R injury seems to involve multiple target pathways such as Akt, KOR and/or DOR signaling.</p></div

The role of different opioid receptors subtype affecting mortality in preconditioned remote electro-stimulation (RES)-induced myocardial protection against ischemia/reperfusion (I/R) injury.

<p>In the I/R model, animals were randomly allocated into 6 groups as described in Methods. They were 1) sham RES group (n = 24); 2) RES preconditioning group (n = 20); 3) RES preconditioning pretreated with KOR blocker (n = 18); 4) RES preconditioning pretreated with DOR blocker (n = 10); 5) RES preconditioning pretreated with KOR/MOR antagonists, which left DOR active (n = 11); 6) RES preconditioning pretreated with DOR/MOR antagonists, which left KOR active (n = 9). The mortality rate (A), duration of tachyarrhythmia (B), CK-MB (C) and troponin I (D) were evaluated during I/R injury. Data are presented as mean ± S.E.M and were analyzed using repeated one-way analysis of variance (ANOVA) followed by the Dunnet’s test. Mortality rate was analyzed using Fisher’s exact test. A <i>p</i> value less than 0.05 is considered statistically significant. *, <i>p</i><0.05, <i>vs</i>. sham RES; #, <i>p</i><0.05, <i>vs</i>. RES. KOR, kappa opioid receptor; DOR, delta opioid receptor; MOR, mu opioid receptor. KOR left, KOR activity remained; DOR left, DOR activity remained; MOR left, MOR activity remained.</p

Molecular evidence of kappa opioid receptor (KOR) in rat heart.

<p>After rat hearts were harvested from the animals, total RNA was isolated and tissue specimen prepared for real-time PCR (A) and immunofluorecent staining (B) as described in Methods. The real-time PCR was analyzed using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as internal control and normalized using mu opioid receptor as relative baseline level. KOR, kappa opioid receptor; DOR, delta opioid receptor; MOR, mu opioid receptor. Yellow arrows indicate the localization of KOR in cardiomyotes while white arrow heads indicate the margin of a vessel. The results were repeated in three independent experiments, only represented figure was shown.</p

The role of different opioid receptors subtype in hemodynamic changes of preconditioned remote electro-stimulation (RES)-induced myocardial protection against ischemia/reperfusion (I/R) injury.

<p>Animals were randomly allocated into 6 groups as described in Methods. They were 1) sham RES group (n = 24); 2) RES preconditioning group (n = 20); 3) RES preconditioning pretreated with KOR blocker (n = 18); 4) RES preconditioning pretreated with DOR blocker (n = 10); 5) RES preconditioning pretreated with KOR/MOR antagonists, which left DOR active (n = 11); 6) RES preconditioning pretreated with DOR/MOR antagonists, which left KOR active (n = 9). During I/R injury period, the hemodynamic changes in these animals were continuously monitored (A). The results showed that preconditioned RES seemed to maintain a higher mean arterial pressure (MAP) than the sham RES group (B). It was of note that there was a significant decreased MAP in preconditioned RES + KOR blocked group (C), but not in preconditioned RES + DOR blocked group (D), compared to the preconditioned RES group. *, <i>p</i><0.05, <i>vs</i>. sham RES. KOR, kappa opioid receptor; DOR, delta opioid receptor; MOR, mu opioid receptor; KOR left, KOR activity remained; DOR left, DOR activity remained; MOR left, MOR activity remained.</p

Study design and protocols.

<p>Two protocols were designed in this study, namely, 1) mechanistic study and 2) I/R model. In mechanistic protocol, various receptors antagonists were pretreated 15 min before remote electro-stimulation (RES). In I/R model, various receptors antagonists were pretreated 15 min before RES preconditioning, followed by 1h-ischemia and 3 h-reperfusion.</p

The role of opioid receptors signaling in remote electro-stimulation (RES)-induced myocardial GSK3 and PKCε expression.

<p>A non-selective opioid receptor antagonist, naloxone (1 mg/kg) (A, B, n = 4–5), and three specific opioid receptor subtypes antagonists targeting the kappa opioid receptors (KORs), delta opioid receptors (DOR)s and mu opioid receptor (MOR) antagonists (C, D, n = 4–5), were used to pretreat the animals for 15 min before RES preconditioning. Next, after RES treatment for 30 min, the heart proteins were analyzed by Western blotting. Optic density (OD) ratio = phosphorylated form divided by the total form. Only the band of GSK3-β was quantified. *, <i>p</i><0.05, <i>vs</i>. sham RES; #, <i>p</i><0.05, <i>vs</i>. RES-30 or RES. KOR, kappa opioid receptor; DOR, delta opioid receptor; MOR, mu opioid receptor; KOR left, KOR activity remained; DOR left, DOR activity remained; MOR left, MOR activity remained.</p

3-MA alleviated MDMA-induced cortical neuron death.

<p>(A) Cortical neuron cultures were treated with different concentrations of MDMA in the presence or absence of 1 mM 3-MA. After 48 h, the neuronal death was determined by LDH cytotoxicity assay. The data are presented as percentage of dead cells. n = 3, triplicate wells for each condition, *p<0.05, **p<0.01 vs control. (B) Cortical neuron cultures were treated with 2 mM MDMA in the presence or absence of 1 mM 3-MA. After 48 h, the cells were immunostained with anti-NeuN antibody. Bar  = 50 µm. (C) Quantitative data of the number of NeuN-positive cells. The data are presented as percentage of the control value (mean ± S.D.). n = 3, ^##<i>P</i><0.01 vs control; **p<0.01 vs 2 mM MDMA treatment. (D) 3-MA attenuated MDMA-induced activation of caspase 3. Cortical neuron cultures were treated with 2 mM MDMA in the presence or absence of 1 mM 3-MA for 24 h, and then western blot analysis was performed using antibodies against cleaved-caspase 3 and caspase 3. β-actin was used as the internal control. Quantitative data are expressed as intensity relative to the control mean value (mean ± S.D.). n = 3, ^#<i>P</i><0.05 vs control; *p<0.05 vs 2 mM MDMA treatment. (E) Representative images from cortical neuron cultures treated with 2 mM MDMA in the presence or absence of 1 mM 3-MA. After 24 h, the apoptotic cells were determined by immunostaining with anti-cleaved caspase 3. Bar  = 30 µm. (F) Quantitative data of the number of cleaved-caspase 3 positive neurons. The data are presented as percentage of the control value (mean ± S.D.). n = 3, ^##<i>P</i><0.01 vs control; **p<0.01 vs 2 mM MDMA treatment.</p

Autophagy Activation Is Involved in 3,4-Methylenedioxymethamphetamine (‘Ecstasy’)—Induced Neurotoxicity in Cultured Cortical Neurons

<div><p>Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.</p></div

The time course of MDMA-induced autophagy activation in cultured cortical neurons.

<p>Cultured cortical neurons were treated with MDMA for different times as indicated, and then subjected to western blot analysis and double immunofluorescence staining with anti-LC3B and MAP2 antibodies. (A) Western blot analysis of LC3B. β-actin was used as the internal control (upper panel). Densitometry of LC3B-II/LC3B-I (bottom panel). Quantitative data are expressed as intensity relative to the control mean value (mean ± S.D.). n = 3, *<i>P</i><0.05 vs control. (B) Representative double immunofluorescence and merged images using anti-LC3B and anti-MAP2 antibodies. Bar  = 30 µm. Intense LC3 punctate staining pattern shown in the MDMA-treated group (see insets). (C) Quantitation of neurite outgrowth. The total neurite length are shown as mean ± S.D., n = 3 experiments with 200–300 cells per experiment, *<i>P</i><0.05 vs control.</p

Dose dependence of MDMA-induced autophagy activation and neurite degeneration in cortical neuron culture.

<p>Cultured cortical neurons were treated with the indicated concentrations of MDMA for 48 h, and then western blot analysis and double immunofluorescence were performed. (A) Western blot analysis of LC3B and beclin-1. β-actin was used as the internal control. (B) Densitometry of LC3B-II/LC3B-I ratio. Quantitative data are expressed as intensity relative to the control mean value (mean ± S.D.). n = 3, *<i>P</i><0.05 vs control. (C) Double immunofluorescence stained images and merged images of representative cells using anti-LC3B and anti-MAP2. Bar  = 30 µm. Intense LC3 punctate fluorescence shown in the MDMA-treated group (see insets). (D) Quantitation of neurite outgrowth. The total neurite length are shown as mean ± S.D., n = 3 experiments with 200–300 cells per experiment, *<i>P</i><0.05 vs control.</p