Objective Cardiac HERG potassium channel currents can cause long QT syndrome .The function of the cardiac HERG potassium channel is regulated by tyrosine kinase and phosphatase , and protein tyrosine phosphatase non-receptor type 11 ( PTPN11) negatively regulates the HERG potassium channel .This study aimed to investigate the effect of PTPN 11 on the electrophysio-logical characteristics of the HERG channel . Methods The plasmids of pcDNA3.1-PTPN11-EGFP were constructed by PCR technique and transfected or cotransfected with the pcDNA 3.1-PTPN11-EGFP plasmid into HEK293 cells using Lipofectamine 2000.The patch clamp technique was employed to record the HERG channel currents in the control group ( HEK293 cells transfected with pcDNA3.0-HERG-EGFP), PTPN11 overexpression group (pcDNA3.0-HERG and pcDNA3.1-PTPN6-EGFP cotransfected HEK293 cells), and PTPN11 overexpression with PAO group . Results The pcDNA3.1-PTPN11-EGFP plasmid was successfully constructed .Green fluorescence was observed in the HEK293 cells transfected with pcDNA3.0-HERG-EG-FP or cotransfected with pcDNA3.0-HERG and pcDNA3.1-PTPN11.The maximum densities of pulse and tail currents were significantly decreased in the PTPN11 overexpression group as compared with the control ([31.85 ±5.54] vs [45.92 ±3.18] pApF, P<0.05;[73.82 ±11.31] vs [108.43 ±7.98] pApF, P<0.05) but markedly in-creased in the PTPN11 overexpression with PAO group ([48.08 ±4.32] pApF;[120.06 ±8.02] pApF) (P<0.05).The time con-stant of deactivation was significantly higher in the PTPN 11 overexpression group than in the control ([622.16 ±46.49] vs [440.70 ± 49.49] ms, P<0.05). Conclusion The overexpression of PTPN11 decreases HERG potassium channel currents , which can be re-versed by PAO.This finding provides a theoretical basis for the application of certain regulatory enzymes in the HERG channel as a treat -ment of long QT syndrome .

AIM: To study the impacts of the myocardial connexin 43 degradation on conduction velocity during acute myocardial ischemia. METHODS: Studies were carried out in 16 dogs which were randomly divided into control group( n= 4) and ischemia group( n= 12). The acute myocardial ischemia was induced by ligation of the left anterior descending coronary artery. The conduction velocity of ischemic myocardium was determined. The content of Cx43 of the ischemic myocardium was examined by laser confocal microscopy with a double-label immunohistochemistry technique. RESULTS: (1)The Cx43 degraded rapidly during acute myocardial ischemia, and the conduction velocity of ischemic myocardium declined greatly; (2)The conduction velocity correlated positively with the Cx43 pixel density in each small region of ischemic myocardium; (3) The Cx43 pixel density decreased over 50% in the region which occuring permanent conduction block. CONCLUSION: These data suggest the degradation of Cx43 decreases the conduction velocity greatly during acute short time myocardial ischemia, and severe degradation of Cx43 would lead to permanent conduction block.

OBJECTIVETo study the effect of protein tyrosine phosphatase non-receptor type 12 (PTPN12) in regulating cardiac HERG channel currents.

METHODSThe plasmids pcDNA3.1-PTPN12-RFP and herg mutant constructed by PCR technique were transfected into HEK293 cells via Lipofectamine 2000, and the cells stably expressing PTPN12 selected with G418 were identified by Western blotting with anti-PTPN12 antibody. HERG channel current in cells expressing HERG alone (HEK293/HERG cells), cells overexpressing PTPN12 (HEK293/HERG cells transfected with pCDNA3.1-PTPN12-RFP), PAO-treated cells (PTPN12/HERG cells treated with PAO), and herg mutant cells (HEK293/HERGY327A-Y700A-Y845A cells transfected with pcDNA3.1-PTPN12-RFP) were recorded by patch-clamp technique.

RESULTSThe plasmids pcDNA3.1-PTPN12-RFP and herg mutant were successfully constructed, and the stable expressing cell lines were established. Red fluorescence was obversed in HEK293/HERG cells transfected with pcDNA3.1-PTPN12-RFP, and the protein expression of PTPN12 was detected. Overexpression of PTPN12 significantly decreased HERG current density in HEK293/HERG cells, and this change was significantly weakened in the inhibitor group and herg mutant group.

CONCLUSIONPTPN12 negatively regulates cardiac HERG channel cerrent possibly by decreasing the phosphorylation level of HERG tyrosine residues. This finding provides further insight into the regulatory mechanism of HERG channel and the pathogenesis of long QT syndrome.

Ether-A-Go-Go Potassium Channels ; physiology ; HEK293 Cells ; Heart ; Humans ; Long QT Syndrome ; Patch-Clamp Techniques ; Protein Tyrosine Phosphatase, Non-Receptor Type 12 ; physiology ; Transfection

Objective: To assess the interobserver variations in delineating the planning target volume (PTV) and organs at risk (OAR) using different contouring methods during intensity-modulated radiation therapy (IMRT) for nasopharyngeal carcinoma (NPC), aiming to provide references for the quality control of multi-center clinical trials. Methods: The PTV and OAR of CT image of 1 NPC patient manually delineated by 10 physicians from 8 different radiation centers were defined as the " manual contour group" , and the OAR auto-contoured using the ABAS software and modified by the physicians were defined as the " auto+ manual contour group" . The maximum/minimum ratio (MMR) of the PTV and OAR volumes, and the coefficient of variation (CV) for different delineated contours were comparatively evaluated. Results: Large variation was observed in the PTV and OAR volumes in the manual contour group. The MMR and CV of the PTV were 1.72-3.41 and 0.16-0.39, with the most significant variation in the PTV_nd (MMR=3.41 and CV=0.39 for the PTV_nd-L). The MMR and CV of the manually contoured OAR were 1.30-7.89 and 0.07-0.67. The MMR of the temporal lobe, spinal cord, temporomandibular joint, optic nerve and pituitary gland exceeded 2.0. Compared with the manual contour group, the average contouring time in the auto+ manual group was shortened by 68% and the interobserver variation of the OAR volume was reduced with an MMR of 1.04-2.44 and CV of 0.01-0.37. Conclusions Large variation may occur in the PTV and OAR contours during IMRT plans for NPC delineated by different clinicians from multiple medical centers. Auto-contouring+ manually modification can reduce the interobserver variation of OAR delineation, whereas the variation in the delineation of small organs remains above 1.5 times. The consistency of the PTV and OAR delineation and the possible impact upon clinical outcomes should be reviewed and evaluated in multi-center clinical trials.