Annexin V staining during reperfusion detects cardiomyocytes with unique properties.
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.
Narayan
P
P
Department of Surgery, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA. pnaraya@pop.uky.edu
Mentzer
R M
RM
Lasley
R D
RD
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
D017304:Annexin A5; D002118:Calcium