After isolation of neonatal and adult cardiomyocytes, they were loaded with a Ca2+ fluorophore (10 µmol/L Fluo‐4 AM, Invitrogen), observed with Leica AF6000 fluorescence microscope, and Ca2+ handling detected as previously described. 27 Briefly, systolic Ca2+ transient was recorded in steady‐state conditions under constant field stimulation (1.0 Hz, 2.0‐ms duration, stimulation voltage set to 1.5 times the threshold). The Ca2+ content of the sarcoplasmic reticulum and the amplitude of caffeine‐induced Ca2+ transient were also recorded. The PKA inhibitor (H89, 10µM, MCE) was administered as previously described. 27 Additional details are provided in the supplementary material online.

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