The vehicle at this concentration had no observable effect on the hearts. In a separate series of experiments, epicardial monophasic action potentials (MAPs) were recorded from the surface of the left ventricle in isolated hearts with control perfusate, and with riluzole added to the perfusate at Doxorubicin
3, 10 and 30??M (n= 6). Data was digitized at 2 KHz using ��Powerlab�� hardware and software from AD Instruments (Sydney, Australia). Measurements of left ventricular pressure (LVP) and the differential of LVP (dLVP/dt) were made during normal perfusion, during ischaemia, and 1?min after reperfusion, by averaging the measured variables over 10 consecutive beats. Action potential duration (APD) measurements were made with the ��peak parameters�� BML-190
add-on module for Chart 5 (AD Instruments) All measurements were made without knowledge of the treatments. Data are presented as means �� standard error of the mean and statistical analysis (Student's t-test) was done in Excel (Microsoft). Significance was taken at P < 0.05. Myocytes were isolated from adult Wistar rat hearts as described previously (McCrossan et?al., 2004). The freshly isolated myocytes were incubated with 2??M Fura 4-AM (Molecular Probes, Eugene, OR, USA) and gently shaken for 20?min at room temperature (20�C24��C). Cells were then resuspended in a HEPES-based solution (see further discussion) and left in the dark for 30?min before use to allow for de-esterification of the dye. Myocytes were placed on the stage of a Nikon Eclipse microscope for simultaneous recording of cell length and [Ca2+]i. Cells were field stimulated at 2?Hz at 120% threshold voltage. For all measurements, cells were superfused continuously at 37��C with a HEPES-based solution containing (in mM): 137 NaCl; 5.4 KCl; 0.33 NaH2PO4; 0.5 MgCl2; 5 HEPES; 5.6 glucose; 1 CaCl2 at pH?7.4. Cell length was monitored using a video-edge detector (Crescent Electronics, Salt Lake City, UT, USA). For the [Ca2+]i measurement, myocytes were alternately illuminated at 340 and 380?nm, using an Optoscan monochromator (Cairn Research, Kent, UK). The fluorescent emission at 510?nm was collected by a photomultiplier tube and the ratio DAPT
of emitted light in response to excitation at 340 and 380?nm was calculated to provide an index of [Ca2+]i. In order to induce hypoxia, the superfusate was bubbled with 100% N2, which was also blown over the surface of the partially closed experimental chamber to maintain a low PO2 (��28?mmHg) in the experimental chamber. Low PO2 was confirmed by measuring samples of the perfusate using a blood gas analyser (Bayer Rapid Lab 348, Siemens Healthcare Diagnostics, Deerfield, IL, USA). Myocytes were exposed to an O2-bubbled solution for 10?min.