Dexmedetomidine Protects Human Cardiomyocytes Against Ischemia-Reperfusion Injury Through α2-Adrenergic Receptor/AMPK-Dependent Autophagy

Abstract

Background: Ischemia-reperfusion injury (I/R) strongly affects the prognosis of children with complicated congenital heart diseases (CHDs) who undergo long-term cardiac surgical processes. Recently, the α2-adrenergic receptor agonist Dexmedetomidine (Dex) has been reported to protect cardiomyocytes (CMs) from I/R in cellular models and adult rodent models. However, whether and how Dex may protect human CMs in young children remains largely unknown. Methods and Results: Human ventricular tissue from tetralogy of Fallot (TOF) patients and CMs derived from human-induced pluripotent stem cells (iPSC-CMs) were used to assess whether and how Dex protects human CMs from I/R. The results showed that when pretreated with Dex, the apoptosis marker-TUNEL and cleaved caspase 3 in the ventricular tissue were significantly reduced. In addition, the autophagy marker LC3II was significantly increased compared with that of the control group. When exposed to the hypoxia/reoxygenation process, iPSC-CMs pretreated with Dex also showed reduced TUNEL and cleaved caspase 3 and increased LC3II. When the autophagy inhibitor (3-methyladenine, 3-MA) was applied to the iPSC-CMs, the protective effect of Dex on the CMs was largely blocked. In addition, when the fusion of autophagosomes with lysosomes was blocked by Bafilomycin A1, the degradation of p62 induced by Dex during the autophagy process was suspended. Moreover, when pretreated with Dex, both the human ventricle and the iPSC-CMs expressed more AMP-activated protein kinase (AMPK) and phospho AMPK (pAMPK) during the I/R process. After AMPK knockout or the use of an α2-adrenergic receptor antagonist-yohimbine, the protection of Dex and its enhancement of autophagy were inhibited. Conclusion: Dex protects young human CMs from I/R injury, and α2-adrenergic receptor/AMPK-dependent autophagy plays an important role during this process. Dex may have a therapeutic effect for children with CHD who undergo long-term cardiac surgical processes.

Keywords: autophagy; cardiomyocyte; congenital heart disease; dexmedetomidine; ischemia-reperfusion injury.

FIGURE 1

Dex protected human ventricular tissue from apoptosis and was associated with autophagy. (A) Timelines of the Dex, cardiopulmonary bypass (CBP), and reperfusion treatments. (B) Representative immunofluorescence images of the ventricular tissues pretreated with Dex and the control tissues. Blue (DAPI), red (TUNEL), and white (cTnT). (C) Quantification of the TUNEL positive cells. N = 6 patients, ten slides/patient. (D) Representative pro-cleaved caspase 3 Wes blot of atrial tissues pretreated with Dex. (E) Quantification of the cleaved caspase 3 relative expression. N = 6 patients. (F) Quantification of the pro-caspase 3 relative expression. N = 6 patients. (G) Representative LC3/p62 Wes blot of the atrial tissues pretreated with Dex. (H) Quantification of the LC3II relative expression. N = 6 patients. (I) Quantification of the p62 relative expression. N = 6 patients.

FIGURE 2

Dex protected human iPSC-CMs from apoptosis and was associated with autophagy. (A) Timelines of the Dex, hypoxia (1% O₂) and reoxygenation (21% O₂) treatments. (B) Representative immunofluorescence images of the iPSC-CM pretreated with Dex. Blue (DAPI), red (TUNEL), and green (sarcomeric α- actin, SAA). (C) Quantification of the TUNEL-positive cells. N= 10 fields from three independent experiments. (D) Representative pro-cleaved caspase 3 Wes blot of the iPSC-CMs pretreated with Dex at the time of post-reoxygenation (12 h). (E) Quantification of the cleaved caspase 3 relative expression. N = 6 replicates. (F) Quantification of the pro- caspase 3 relative expression. N = 6 replicates. (G) Representative LC3/p62 Wes blot of the iPSC-CMs pretreated with Dex at the time of post-reoxygenation (12 h). (H) Quantification of the LC3II relative expression. N = 6 replicates. (I) Quantification of the p62 relative expression. N = 6 replicates.

FIGURE 3

Autophagy inhibitor (3-methyladenine, 3-MA) blocked the autophagy flux induced by Dex. (A) Representative LC3II Wes blots of iPSC-CMs pretreated with Dex and 3-MA. (B) Quantification of the LC3II relative expression. N = 6 replicates. (C) Representative p62 Wes blots of iPSC-CMs pretreated with Dex and 3-MA. (D) Quantification of the p62 relative expression. N = 6 replicates. ** p <0.01. vs. H/R; ## p <0.01, vs. H/R + Dex.

FIGURE 4

The autophagy inhibitor (3-methyladenine, 3-MA) blocked the protection of Dex during I/R injury. (A) Representative TUNEL immunofluorescence images of iPSC-CMs pretreated with 3-methyladenine (3-MA). Blue (DAPI), green (TUNEL), red (sarcometric α-actin, SAA). (B) Quantification of the TUNEL- positive cells. N= 10 fields from three independent experiments. (C) Representative pro-cleaved caspase 3 Wes blot of iPSC-CM pretreated with Dex and 3-MA. (D) Quantification of the pro-caspase 3 relative expression. N = 6 replicates. (E) Quantification of the cleaved caspase 3 relative expression. N= 6 replicates.

FIGURE 5

Dex activated autophagy but did not block autophagosomal maturation. (A) Representative LC3II Wes blot of iPSC-CM pretreated with Dex and BafA1 (the inhibitors of autophagosomal maturation). (B) Quantification of the LC3II relative expression. N= 6 replicates. (C) Representative p62 Wes blot of the iPSC-CM pretreated with Dex and BafA1. (D) Quantification of the p62 relative expression. N= 6 replicates. * p <0.05, ** p <0.01. vs. H/R; ## p <0.01, vs. H/R + Dex.

FIGURE 6

The autophagy induced by Dex was AMPK dependent. (A) Representative AMPK/p-AMPK Wes blot of a human ventricle pretreated with Dex. (B) Quantification of the AMPK relative expression of a human atria. N = 6 patients. (C) Quantification of the p-AMPK relative expression of a human ventricle. N = 6 patients. (D) Representative AMPK/p-AMPK Wes blot of iPSC-CMs pretreated with Dex. (E) Quantification of the AMPK relative expression of iPSC-CMs. N = 6 replicates. (F) Quantification of the p-AMPK relative expression of iPSC-CMs. N = 6 replicates. (G) The expression of AMPK and p-AMPK were reduced by AMPK siRNA, as indicated by the Wes blot. (H) The expression of p-AMPK was reduced by AMPK siRNA, as indicated by immunostaining. (I) AMPK siRNA blocked the increased expression of LC3II by Dex, as indicated by the Wes blot. (J) Quantification of the LC3II expression in Panel 6I. (K) AMPK siRNA blocked the reduced expression of p62 by Dex, as indicated by the Wes blot. (L) Quantification of the p62 expression in Panel 6K. ** p <0.01. vs. H/R; ## p <0.01, vs. H/R + Dex.

FIGURE 7

The protection of Dex was α2-adrenergic receptor dependent. (A) Representative LC3II Wes blot of iPSC-CMs pretreated with Dex and yohimbine (α2-adrenergic receptor antagonist). (B) Quantification of the LC3II relative expression. N = 3 independent experiments. (C) Representative p62 Wes blot of iPSC-CMs pretreated with Dex and yohimbine. (D) Quantification of the p62 relative expression. N = 3 independent experiments. (E) Representative TUNEL-positive CMs after treatment with Dex and yohimbine during the H/R process. (F) Quantification of the TUNEL-positive CMs. N = 10 fields. (G) Representative pro-cleaved caspase 3 Wes blot of iPSC-CMs pretreated with Dex and yohimbine. (H) Quantification of the pro-caspase 3 relative expression. N = 6 replicates. (I) Quantification of the cleaved caspase 3 relative expression. N = 6 replicates. ** p <0.01. vs. H/R; ## p <0.01, vs. H/R + Dex.