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.