title: Small interference RNA against PTP-1B reduces hypoxia/reoxygenation induced apoptosis of rat cardiomyocytes.
abstract: OBJECTIVE
  To investigate the effect of siRNA against PTP-1B on neonatal rat cardiac myocyte apoptosis induced by hypoxia-reoxygenation (H/R) and its molecular mechanisms.
  
  
  METHODS
  Isolated neonatal and adult rat cardiac myocytes were cultured for 24 h after PTP-1B siRNA transfection, and with 2, 4 and 6 h of hypoxia followed by 6 h of reoxygenation (H/R). The cardiac myocyte apoptosis induced by the treatments was assessed by TUNEL staining. Levels of PTP-1B and phospho-Akt were determined by Western blot, colorimetric assay kits were used to measure activities of caspase-3 and 8, and co-immunoprecipitation was used to check the amount of PTP-1B bound to FasR. Sodium orthovanadate, a general pharmacological phosphatase blocker and LY294002, an inhibitor of PI3-kinase/Akt pathway, were respectively used to inhibit PTP-1B and Akt activity.
  
  
  RESULTS
  H/R resulted in severe injury in cultured rat cardiomyocytes and upregulated PTP-1B expression. However, siRNA against PTP-1B significantly decreased the number of apoptotic cardiomyocytes induced by 4H/6R as compared with cells without siRNA treatment (Apoptotic index: 12.1 +/- 1.4% vs. 23.2 +/- 1.6%, P < 0.05), along with greater phosphorylation of Akt, reduced activities of caspase-3 and 8, and the lower association of PTP-1B with FasR. Vanadate and LY294002 also partly reduced apoptosis of cardiomyocytes induced by 4H/6R.
  
  
  CONCLUSIONS
  PTP-1B is a key regulator of apoptosis of cardiomyocytes induced by H/R, and siRNA against PTP-1B effectively protects cardiomyocytes against H/R injury, the mechanisms of which might be associated with Akt activation, the reduction of both caspase-3 and 8 activities, and the lower amount of PTP-1B bound to FasR.
authors.affiliation: Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Huiwen
authors.initials: H
authors.lastname: Song
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Zhang
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
mesh_terms: D000375:Aging; D000818:Animals; D017209:Apoptosis; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D000860:Hypoxia / Q000503:physiopathology*; D020287:In Situ Nick-End Labeling; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D015428:Myocardial Reperfusion Injury; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015854:Up-Regulation; D014638:Vanadates / Q000494:pharmacology
chemical_list: D061945:Caspase Inhibitors; D002867:Chromones; D009025:Morpholines; D034741:RNA, Small Interfering; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014638:Vanadates; D051057:Proto-Oncogene Proteins c-akt; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; C516610:Ptpn1 protein, rat