Reduction of Mitochondria–Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury
Objective—We explored the role of endoplasmic reticulum (ER)–mitochondria Ca2+ cross talk involving voltage-dependent anion channel-1 (VDAC1)/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 in endothelial cells during hypoxia/reoxygenation (H/R), and investigated the protective effects of acetylcholine.
Approach and Results—Acetylcholine treatment during reoxygenation prevented intracellular and mitochondrial Ca2+ increases and alleviated ER Ca2+ depletion during H/R in human umbilical vein endothelial cells. Consequently, acetylcholine enhanced mitochondrial membrane potential and inhibited proapoptotic cascades, thereby reducing cell death and preserving endothelial ultrastructure. This effect was likely mediated by the type-3 muscarinic acetylcholine receptor and the phosphatidylinositol 3-kinase/Akt pathway. In addition, interactions among members of the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex were increased after H/R and were associated with mitochondrial Ca2+ overload and cell death. Inhibition of the partner of the Ca2+ channeling complex (VDAC1 siRNA) or a reduction in ER–mitochondria tethering (mitofusin 2 siRNA) prevented the increased protein interaction within the complex and reduced mitochondrial Ca2+ accumulation and subsequent endothelial cell death after H/R. Intriguingly, acetylcholine could modulate ER–mitochondria Ca2+ cross talk by inhibiting the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 expression. Phosphatidylinositol 3-kinase siRNA diminished acetylcholine-mediated inhibition of mitochondrial Ca2+ overload and VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex formation induced by H/R.
Conclusions—Our data suggest that ER–mitochondria interplay plays an important role in reperfusion injury in the endothelium and may be a novel molecular target for endothelial protection. Acetylcholine attenuates both intracellular and mitochondrial Ca2+ overload and protects endothelial cells from H/R injury, presumably by disrupting the ER–mitochondria interaction.
- Received July 25, 2014.
- Accepted April 28, 2015.
- © 2015 American Heart Association, Inc.