HL-1 cardiomyocyte contractility and Ca2+ flux were recorded using a video edge-recognition system (IonOptix, MyoCam-S, Dublin, Ireland), as previously described [39 (link),79 (link)]. Briefly, 106 cells were seeded into 35 mm glass-bottom tissue culture dishes (Corning) and cultured at 37°C and 5% CO2 until approximately >75% of the cells were spontaneously contracting (2–3 days). Experiments were performed on spontaneously contracting as well as on paced cardiomyocytes (at 5 Hz) and comparable results were obtained. Cells that showed irregular contractions or no response to electrical stimulation were excluded. After one minute of recording, treatment was gently perfused across the cells and contractility was recorded for a period of 30 min. In experiments with chemical inhibitors (Go6976 or LY333531 (Sigma) or 4-Phenylbutyric acid (4-PBA, Calbiochem), these were pre-incubated with the cells for 1 h prior to PLY treatment. All recordings were performed at 37°C. Mechanical parameters of contractility (Peak Shortening, TTP, tR90, +dL/dt and -dL/dt) were analyzed using the Ion-Wizard 6.0 software.
For Ca2+ flux recording, Fura-2AM (Invitrogen) (10 μM) was incubated with cells for 45 min, washed and incubated at 37°C for a further 10 min in fully supplemented Claycomb medium to allow the dye to de-esterify. Cells were then mounted into an inverted microscope and intracellular calcium flux was recorded continuously using an Optoscan monochromator fluorescence photometry (Cairn Research, Faversham, Kent, UK) for 30 min after treatment. Fluorescence signals were elicited by alternate excitations with respective wavelengths of 340 and 380 nm at 250 Hz and recorded at 510 nm through a photomultiplier tube. In order to quantify [Ca2+]i, cells were treated with (10 mM) Caffeine (Sigma,UK) to get the maximum (Rmax) and (25 mM) EDTA to get the minimum (Rmin) intensity. [Ca2+]i = Kd*(R-Rmin)/(Rmax-R)*Sf2/Sb2, where Kd is the Fura-2AM dissociation constant (set at 225 nM). The values of Sb2 and Sf2 correspond to fluorescence excited by the denominator wavelength under conditions of saturating calcium levels (bound state) and in the absence of calcium (calcium-free state) respectively.
For Ca2+ flux recording, Fura-2AM (Invitrogen) (10 μM) was incubated with cells for 45 min, washed and incubated at 37°C for a further 10 min in fully supplemented Claycomb medium to allow the dye to de-esterify. Cells were then mounted into an inverted microscope and intracellular calcium flux was recorded continuously using an Optoscan monochromator fluorescence photometry (Cairn Research, Faversham, Kent, UK) for 30 min after treatment. Fluorescence signals were elicited by alternate excitations with respective wavelengths of 340 and 380 nm at 250 Hz and recorded at 510 nm through a photomultiplier tube. In order to quantify [Ca2+]i, cells were treated with (10 mM) Caffeine (Sigma,UK) to get the maximum (Rmax) and (25 mM) EDTA to get the minimum (Rmin) intensity. [Ca2+]i = Kd*(R-Rmin)/(Rmax-R)*Sf2/Sb2, where Kd is the Fura-2AM dissociation constant (set at 225 nM). The values of Sb2 and Sf2 correspond to fluorescence excited by the denominator wavelength under conditions of saturating calcium levels (bound state) and in the absence of calcium (calcium-free state) respectively.
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