Paper BM-TuP4
Electrical Assay for Real-Time Monitoring Cardiomyocyte Apoptosis

Tuesday, October 21, 2008, 6:30 pm, Room Hall D

Deregulated cardiomyocyte apoptosis is a critical risk factor in a variety of cardiovascular diseases. Though enzymatic DNA fragmentation is most commonly used criteria of apoptosis at the level of individual cardiomyocytes, the capability of detecting cell detachment will provide instant information at early phase of apoptosis. Furthermore, the assays used to detect DNA fragmentation are all invasive to living cells, which disables real-time monitoring of the whole process. In this work, we developed an impedance-sensing assay for real time monitoring cardiomyocyte apoptosis induced by tumor necrosis factor alpha (TNF-alpha) based on recording the change in cardiomyocyte adhesion to extracellular matrix (ECM). Electrochemical impedance spectroscopy (EIS) was employed in impedance to process the impedance spectra, followed by manual calibration with electrical cell-substrate impedance sensing (ECIS) technique. Adhesion profile of cardiomyocytes undergoing cell death process was recorded in a time course of equivalent cell-substrate distance. Multiple concentration levels of TNF-alpha (from 10 to 80 ng/mL) were applied to the cultured cardiomyocytes and the concentration-related adhesion profiles were recorded for the cell death process. An optimal concentration of TNF-alpha (20 ng/mL) was determined to induce cardiomyocyte apoptosis rather than necrosis because of its mild slope of developing cell detachment in 24-hour real-time monitoring. It was also observed in the Trypan blue exclusion (TBE) results that a gradual and significant increment in cell death rate was achieved with a concentration level of 20 ng/mL. Treat with optimal concentration of TNF-alpha, the cardiomyocytes first experienced a transient drop in cell-substrate distance followed a sustained cell detachment. The equivalent cell-substrate distance increased from 59.1 to 89.2 nm within 24 hours. The early change of cell adhesion was proven related to cardiomyocyte apoptosis with the following TUNEL test in which the treated cardiomyocytes suffered an apoptotic percentage of 21.1 ± 5.5 % (vs. 5.9 ± 2.5 % in the control sample). This novel assay has the potential to become a valuable high-throughput experimental approach in studying in vitro cardiomyocyte apoptosis research.