Cardiomyocyte Viability and Contractility Evaluation

Cardiomyocyte viability and contractility were evaluated at three time points throughout the experimental protocol: 7 hours after cell isolation, after transfection with siRNA (before ischemia), and at the end of reperfusion (Figure 1). The viability of cardiomyocytes was assessed by the trypan blue exclusion test [29 (link)–31 (link)]. Cardiomyocyte contractility was measured using the IonOptix system and IonWizard 6.0 software (IonOptix, Milton, MA USA). After a stabilization period the chamber containing the cells was perfused with an oxygenated buffer at a constant temperature of 37°C. Cells were continuously paced (stimulated) at 1 Hz and 5 V (IonOptix MyoPacer, Milton, MA, USA). The assessment of cardiomyocyte contractility was made by the measurement of peak shortening, maximal velocity of cell shortening, and maximal velocity of cell relengthening [21 (link)] on 8–10 cardiomyocytes per independent experiment, over a 10 min period to give an average measure per sample.