ABSTRACT
The effects of long-term nitrate therapy are compromised due to protein S-Nitrosylation, which is mediated by nitric oxide (NO). This study is to determine the role of Akt S-Nitrosylation in the recovery of heart functions after ischaemia. In recombinant Akt protein and in HEK293 cells, NO donor decreased Akt activity and induced Akt S-Nitrosylation, but was abolished if Akt protein was mutated by replacing cysteine 296/344 with alanine (Akt-C296/344A). In endothelial cells, NO induced Akt S-Nitrosylation, reduced Akt activity and damaged multiple cellular functions including proliferation, migration and tube formation. These alterations were ablated if cells expressed Akt-C296/344A mutant. In Apoe-/- mice, nitroglycerine infusion increased both Akt S-Nitrosylation and infarct size, reduced Akt activity and capillary density, and delayed the recovery of cardiac function in ischaemic hearts, compared with mice infused with vehicle. Importantly, these in vivo effects of nitroglycerine in Apoe-/- mice were remarkably prevented by adenovirus-mediated enforced expression of Akt-C296/344A mutant. In conclusion, long-term usage of organic nitrate may inactivate Akt to delay ischaemia-induced revascularization and the recovery of cardiac function through NO-mediated S-Nitrosylation.
Subject(s)
Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Myocardial Revascularization , Nitrates/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Adenoviridae/metabolism , Amino Acid Sequence , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Cysteine/metabolism , HEK293 Cells , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice , Mutation/genetics , Neovascularization, Physiologic/drug effects , Nitric Oxide/metabolism , Nitroglycerin/pharmacology , Nitroprusside/pharmacology , NitrosationABSTRACT
OBJECTIVE: To observe the effect of electroacupuncture (EA) preconditioning on cell apoptosis and the content of mitochondrial reactive oxygen species (ROS) in rats with myocardial ischemia/reperfusion injury (MIRI), so as to explore their mechanisms underlying improvement of MIRI. METHODS: Eighty male Wistar rats were randomly divided into control, sham, ischemia reperfusion (IR) and EA groups, with 20 rats in each group. Rats of the control, sham and IR groups were just banded on the boards for 30 min, once daily for 7 days. Before modeling, EA (2 Hz, 1 mA) was applied to "Neiguan" (PC6), "Zusanli" (ST36), "Guanyuan"(CV4) for 20 min, once daily for 7 successive days in the EA group. The MIRI model was established by occlusion of the left anterior descending branch of the left coronary artery for 20 min, followed by reperfusion for 30 min. Ventricular arrhythmia (VA) score was used to evaluate arrhythmia. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of CK-MB. DHE staining was used to detect the content of reactive oxygen species (ROS). The gene expression levels of cytochrome C (Cyt-C), Caspase-9 and Caspase-3 were detected by real-time fluorescent quantitative PCR. RESULTS: Compared with the sham group, VA score, serum CK-MB content, ROS content in heart tissue and Cyt-C, Caspase-9 and Caspase-3 gene expression were significantly up-regulated in the MIRI group (all P<0.01). Following the intervention, the increased VA score, serum CK-MB content, ROS content in heart tissue, Cyt-C, Caspase-9 and Caspase-3 gene expression were all reversed in the EA group compared with the MIRI group (all P<0.01). CONCLUSION: The protective effect of EA preconditioning on MIRI may be based on the regulation of ROS mediated-apoptosis pathway.
