Fluo 3

[Ca²⁺]_i was measured in cell pairs that were AM-loaded with Fluo3 (10 μM; Molecular Probes), excited at 488 nm, and with emitted fluorescence collected at >505 nm. After subtracting background fluorescence, Fluo3 time course signal was normalized to baseline (F/F₀).

Intracellular Ca²⁺ cycling activity in isolated rabbit ventricular myocytes was monitored using a Leica SP5 confocal laser scanning system with a 60 × 1.4 NA oil-immersion objective in XT mode using Ca²⁺-sensitive indicators Fluo-3 (Molecular Probes, Carlsbad, CA, United States), respectively. Cells were loaded with Fluo-3 for 10 min, and after 20 min de-esterification, the dye was excited at 488 nm with an argon laser. Emission was collected at 500–600 nm. CMs were studied in Tyrode solution (see above). Myocytes were paced via field stimulation at 1 Hz using extracellular platinum electrodes. To assess the SR Ca²⁺ load and decay kinetics, 10 mM caffeine was applied at the end of the experiments.
For triggered activity, myocytes were paced at 1 Hz for 30 s, and the latency between the last stimulus in the pacing train and the first SCW was calculated. To assess the effect of mitochondria-derived ROS on CMs from aged rabbits, myocytes were pretreated with the mitochondria-specific ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (mito-TEMPO, 25 μM, Enzo Life Sciences, Farmingdale, NY, United States) for 10 min. The intracellular Ca²⁺ cycling measurements were performed under β-adrenergic stimulation with 100 nM ISO for HL-1 CMs and 30 nM ISO for aged rabbit CMs.

Glucose-high Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), glucose-free DMEM and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA); poly-L-lysine and H₂O₂ were from Sigma-Aldrich (Milan, Italy); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1), Fluo-3, AM and Annexin V-FITC/propidium iodide (PI) were obtained from Molecular Probes, Invitrogen (Milan, Italy), and MTS was from Promega Corp. (Madison, WI, USA); Hank’s solution and trypsin were obtained from HyClone (Logan, UT, USA), and Hoechst 33342, phosphate-buffered saline (PBS) and penicillin/streptomycin (Pen/Strep) were from Beyotime (Shanghai, China). The lactate dehydrogenase (LDH) assay kit was purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA).

Living-cell microscopy to detect depolarization-dependent calcium influx was performed as previously described^{29 (link)}. A confocal laser system (Zeiss LSM 880) and the fluorescent calcium indicator Fluo-3 (Molecular Probes) were used to measure the intracellular free calcium concentration. Cells were loaded with the Ca²⁺-sensitive fluorescence indicator Fluo-3/AM (3 μM; Biotium) at 37 °C for 60 min in a Ringer buffer containing (in mM) 120 NaCl, 2.5 KCl, 20.0 Hepes (pH 7.4), and 11.0 glucose, and depolarized with high-KCl (100 mM) in Ringer solution as previously described. Fluorescence was measured every 0.25 s for a total of 12 s.