Real-time imaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart.
We report a novel real-time imaging model to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, using fluorescent labeled annexin-V. Annexin-V binds to externalized phosphatidylserine (PS) of cells undergoing programmed cell death. With high-magnification (x100-160) real-time imaging, we visualized the binding of annexin-V to single cardiomyocytes. Kinetic studies at the single-cell level revealed that cardiomyocytes started to bind annexin-V within minutes after reperfusion, following an ischemic period of 30 minutes. The amount of bound annexin-V increased rapidly and reached a maximum within 20-25 minutes. Caspase inhibitors decreased the number of annexin-V-positive cardiomyocytes and slowed down the rate of PS exposure of cardiomyocytes that still bound annexin-V. This technology to study cell biology in the natural environment will enhance knowledge of intracellular signaling pathways relevant for cell-death regulation and strategies to manipulate these pathways for therapeutic effect.
Dumont
E A
EA
Cardiovascular Research Institute Maastricht, Universiteitssingel 50, Maastricht, the Netherlands.
Reutelingsperger
C P
CP
Smits
J F
JF
Daemen
M J
MJ
Doevendans
P A
PA
Wellens
H J
HJ
Hofstra
L
L
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D002462:Cell Membrane / Q000473:pathology*; D005456:Fluorescent Dyes / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D015428:Myocardial Reperfusion Injury / Q000473:pathology*; D009206:Myocardium / Q000473:pathology*; D011485:Protein Binding
D017304:Annexin A5; D005456:Fluorescent Dyes