Phosphomimetic Modulation of eNOS Improves Myocardial Reperfusion and Mimics Cardiac Postconditioning in Mice

Objective: Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection. Methods and Results: We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation. Conclusions and Significance: Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.American Heart Association (Predoctoral Fellowship)National Institutes of Health (U.S.) (R01 NS33335)National Institutes of Health (U.S.) (R01 HL57818

Episomal expression of wild-type CFTR corrects cAMP-dependent chloride transport in respiratory epithelial cells

The isolation of the gene responsible for the Cl- ion transport defect in cystic fibrosis (CF) has provided important information about the relationship between the disease pathology and the underlying genetic and biochemical mechanisms. In addition, new areas of investigation and therapy are now possible. Most notably, the isolation of the CF gene, the cystic fibrosis transmembrane conductance regulator (CFTR) has led to the development of different gene therapy strategies. To circumvent possible complications due to insertional mutagenesis and virally induced immune responses, we have employed Epstein-Barr virus (EBV)-based expression vectors for correction of the cAMP-dependent Cl- transport defect associated with CF. A CFTR-containing expression construct under the regulation of the Rous sarcoma virus (RSV) long terminal repeat (LTR) (pREP5/CFTR) was introduced into transformed human airway epithelial cells defective in cAMP-dependent Cl- transport. Transfected cells were assayed for Cl- transport. Transfected cells were assayed for Cl- ion transport by 36Cl- efflux, SPQ, and patch clamp and showed restoration of intact cAMP-dependent transport. CFTR transcription from pREP5/CFTR was detected by Northern hybridization. The level of response to agonists appeared to be dependent on the level of CFTR expression. When cells were tested for functional expression of CFTR after removal of selection pressure, they showed a continuous decrease in responsiveness to forskolin as a function of time after removal of selection. This decreased correlated with a loss of CFTR mRNA and in the loss of CFTR mRNA and in the loss of the pREP5/CFTR. After 12 to 15 weeks growth without selection both cAMP-dependent Cl- transport and plasmid-derived CFTR mRNA were not detectable. However, it was still possible to rescue cAMP-dependent Cl- transport in these transfected cells by reselection suggesting the presence of the CFTR containing plasmid in a portion of the cells. Analysis of DNA indicated that the pREP5/CFTR vector copy number was reduced from 30 copies per cell with continuous selection, to approximately 0.3 copies per cell after 20 weeks without hygromycin B.link_to_subscribed_fulltex