title: Annexin V staining during reperfusion detects cardiomyocytes with unique properties.
abstract: With the use of markers of sarcolemmal membrane permeability, cardiomyocyte models of ischemic injury have primarily addressed necrotic death during ischemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated reperfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programmed cell death. Propidium iodide is a marker of cell necrosis. Under baseline conditions, <1% of cardiomyocytes stained positive for annexin V. After 20 or 60 min of simulated ischemia, there was no increase in annexin V staining, although 60-min simulated ischemia resulted in significant propidium iodide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positive for annexin V. Annexin V-positive cells retained both rod-shaped morphology and contractile function but exhibited the decreased cell width indicative of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and further elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in approximately 3% of myocytes, which had a rounded appearance and membrane blebs. These results suggest that the use of annexin V after simulated ischemia-reperfusion uncovers a population of cardiomyocytes whose characteristics appear to be consistent with cells undergoing apoptosis.
authors.affiliation: Department of Surgery, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA. pnaraya@pop.uky.edu
authors.forename: P
authors.initials: P
authors.lastname: Narayan
authors.forename: R M
authors.initials: RM
authors.lastname: Mentzer
authors.forename: R D
authors.initials: RD
authors.lastname: Lasley
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000032:analysis*; D017209:Apoptosis; D002118:Calcium / Q000032:analysis; D006352:Heart Ventricles / Q000737:chemistry / Q000166:cytology; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000473:pathology; D015428:Myocardial Reperfusion Injury / Q000473:pathology*; D009206:Myocardium / Q000737:chemistry / Q000473:pathology*; D009336:Necrosis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling
chemical_list: D017304:Annexin A5; D002118:Calcium