For isometric force recordings, papillary muscles were mounted in an organ chamber and connected to the force transducer (Scientific Instruments, Heidelberg, Germany). Papillary muscles were superfused with Krebs–Henseleit solution (in mmol/L: NaCl
2 116, KCL 5, NaH
2PO
4 2, MgCl
2 1.2, Na
2SO
4 1.2, NaHCO
3 20, CaCl
2 initially 0.25; end concentration 1.25, glucose 10) that was also oxygenated with 95% O
2 and 5% CO
2 (37°C). Isometric contractions were elicited using electrical field stimulation with a basal stimulation frequency of 4 Hz (voltage 25% above threshold, normally 3–5 V, stimulator, Scientific Instruments, Heidelberg, Germany). Ca
2+ (0.25 mmol/L) was added every 2 min after a 30 min washout phase until the final concentration of 1.25 mmol/L was reached in order to prevent a Ca
2+-induced contracture. After an equilibration period of 20 min, the muscles were gradually stretched until the maximum steady-state twitch force was achieved and 5 μmol/L ranolazine (Ran, Gilead, Palo Alto, USA) or vehicle was added to the bath solution. After having characterized the basal effect of late
I
Na inhibition, force–frequency relationships were obtained using increasingly higher stimulation rates of 2, 4, 6, 8, 10, and then back to 4 Hz. To measure SR Ca
2+-load, post-rest behavior was assessed by using increasing rest intervals of 5 and 10 s between beats at a basal stimulation frequency of 4 Hz [
24,
26]. Short periods of rest increase force of contraction of the first beat upon restimulation which is considered to be dependent on SR Ca
2+-uptake and release [
25]. In addition, persistent premature arrhythmogenic contractions (PACs) were assessed [
26]. PACs had to be stable and to persist continuously over 5 min before the effect of Ran was evaluated [
26].