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 i schemia. In the present study, we used annexin V-propidium iodide staining to examine apoptosis and necrosis after simulated ischemia and simulated re perfusion in rat ventricular myocytes. Annexin V binds phosphatidylserine, a phosphoaminolipid thought to be externalized during apoptosis or programm ed cell death. Propidium iodide is a marker of cell necrosis. Under baselin e conditions, <1% of cardiomyocytes stained positive for annexin V. After 2 0 or 60 min of simulated ischemia, there was no increase in annexin V stain ing, although 60-min simulated ischemia resulted in significant propidium i odide staining. Twenty minutes of simulated ischemia, followed by 20 or 60 min of simulated reperfusion, resulted in 8-10% of myocytes staining positi ve for annexin V. Annexin V-positive cells retained both rod-shaped morphol ogy and contractile function but exhibited the decreased cell width indicat ive of cell shrinkage. Baseline mitochondrial free Ca2+ (111 +/- 14 nM) was elevated in reperfused annexin V-negative cells (214 +/- 22 nM), and furth er elevated in annexin V-positive myocytes (382 +/- 9 nM). After 60 min of simulated reperfusion, caspase-3-like activity was observed in <similar to> 3% of myocytes, which had a rounded appearance and membrane blebs. These re sults suggest that the use of annexin V after simulated ischemia-reperfusio n uncovers a population of cardiomyocytes whose characteristics appear to b e consistent with cells undergoing apoptosis.