Detection of DNA fragmentation in a single apoptotic cardiomyocyte by electrophoresis on a microfluidic device.
The detection of doxorubicin-induced apoptosis in individual cardiomyocytes was performed on a microfluidic device. Microstructures integrated on a CD-like plastic disk were adapted for the selection of individual cells their lysis in an alkaline environment and the separation of released apoptotic DNA fragments. The fragments with typical 180 base pairs ladder pattern were electrophoretically resolved in a 2% solution of linear polyacrylamide with 0.1 M NaOH on a migration distance of 6 mm. The laser-induced fluorescence of fragments labeled by ethidium bromide was monitored by a photomultiplier tube mounted on a confocal microscope. The causal relation between the enhanced doxorubicin concentration and the extent of DNA fragmentation in a single cell was confirmed. The results show that the extent of DNA fragmentation is proportional to the time of a cell treatment. Onset of necrosis was evident in cardiomyocytes treated by doxorubicin for more than 24 h. The adverse effect of doxorubicin, an important cytostatics used for the treatment of many solid tumors, leads to the destruction of cardiomyocytes and, consequently, may result in the heart failure of treated individuals. Therefore, the monitoring of the extent of apoptotic DNA damage of cardiac myocytes represents critical step toward understanding of the development of chronic doxorubicin-induced cardiomyopathy.
Klepárník
Karel
K
Institute of Analytical Chemistry Academy of Sciences of the Czech Republic. klep@iach.cz
Horký
Marcel
M
D000818:Animals; D017209:Apoptosis; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods; D044085:Microfluidics / Q000379:methods*; D032342:Muscle Cells / Q000166:cytology*; D009206:Myocardium / Q000166:cytology*; D051381:Rats
D004247:DNA