Field stimulator

Cell contractility was evaluated as described previously.^{20 (link)} Briefly, the isolated cells
were placed in a chamber with a glass coverslip base mounted onto the stage of
an inverted microscope (Nikon Eclipse, TS100). The chamber was perfused with
HEPES-Tyrode solution plus 10 µM CaCl₂ at
37ºC. Steady-state 1-Hz contractions were elicited via platinum bath electrodes
(Myopacer, Field Stimulator, IonOptix) with 5-ms voltage pulses and an intensity
of 40 V. The cells were visualized on a personal computer monitor with a NTSC
camera (MyoCam, IonOptix) in partial scanning mode. The image was used to
measure cell shortening (our index of contractility) in response to electrical
stimulation using a video motion edge detector (IonWizard, IonOptix). The cell
image was sampled at 240 Hz. Cell shortening was calculated from the output of
the edge detector using an A/D converter (IonOptix, Milton, MA). Cell shortening
(expressed as percentage of resting cell length) and the velocities of
shortening and relaxation were calculated.

Cardiomyocyte cell shortening (CS) and intracellular Ca²⁺ transients (CaT) were measured using Fura-2 AM as described previously [6 (link), 24 (link), 25 (link)]. Briefly, cells were stimulated electrically by a field stimulator (IonOptix, MA) at a frequency of 0.5 Hz. CaT and CS amplitudes reached the steady state within 30 sec after start of pacing stimulation. Therefore, we recorded CaT and CS from 30 sec to 40 sec after start of pacing at the rate of 0.5 Hz. We defined the values of CaT peak and CS peak, which were calculated from averaging 10 consecutive steady CaT waveforms and 10 CS waveforms by using IonOptix analysis software, as the peak CaT and the peak CS of each cardiomyocyte. Ca²⁺-induced fluorescence at 505 nm was measured by excitation at 340 and 380 nm using a dual-excitation spectrofluorometer. The intracellular calcium concentration was calculated as the ratio of the fluorescence emission intensities at these 2 excitation wavelengths [6 (link), 24 (link), 25 (link)].
To determine the dose-dependent effect of landiolol on CS in isolated normal and failing cardiomyocytes, we measured CS with various doses of landiolol (from 0 nM to 1000 nM).

Cells were incubated in a 2 mM Ca²⁺ Ringer’s solution containing 2.5 µM Fluo-4 and 0.1% Pluronic acid for 30 min. Cells were then moved to a Fluo-4–free Ringer’s solution to deesterify the Fluo-4 for 30 min. Following deesterification, Fluo-4–loaded cells were excited with a 488-nm laser and the resulting fluorescence monitored using an inverted microscope with a Plan-Apochromat 63×/1.40 oil objective, connected to an Andor W1 spinning-disk confocal with a Photometrics Prime 95B camera. Images were acquired every 50 ms for a total of 50 s in a 2 mM Ca²⁺ Ringer’s solution at RT using Micromanager (1.4.21) software. Electrical stimulation at 40 V was performed at 1 Hz for 5 s with a Field Stimulator (IonOptix). Intracellular Ca²⁺ activity was measured as Ca²⁺ peak frequency and Ca²⁺ peak amplitude in a Region of Interest (ROI) localized in the soma or proximal neuronal dendrites using ImageJ or ClamPfit software.