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T-tubules (TT) are invaginations of the surface sarcolemma (SS) that mediate the rapid propagation of the action potential (AP) to the cardiomyocyte core. We employed the advantages of an ultrafast random access multi-photon (RAMP) microscope (Sacconi et al., PNAS 2012) with a double staining approach to optically record t-tubular AP and, simultaneously, the corresponding local Ca2+-release in different positions across the cardiomyocytes. Despite a uniform AP between SS and TT at steady-state stimulation, in control cardiomyocytes we observed a non-negligible be variability of local Ca2+-transient amplitude and kinetics. This variability was significantly reduced by applying 0.1μM Isoproterenol, which increases the opening probability of Ca2+-release units. In the rat heart failure model (HF), we previously demonstrated that some tubular elements fail to propagate AP. We found that the tubules unable to propagate AP, displayed a reduced correspondent Ca2+-transient amplitude as well as a slower Ca2+ rise compared to electrically coupled tubules. Moreover variability of Ca2+-transient kinetics were increased in HF. Finally, TT that did not show AP, occasionally exhibited spontaneous depolarizations that were never accompanied by local Ca2+-release in the absence of any pro-arrhythmogenic stimulation. Simultaneous recording of AP and Ca2+-transient allows us to probe the spatio-temporal variability of Ca2+-release, whereas the investigation of Ca2+-transient in HF discloses an unexpected uncoupling between t-tubular depolarization and Ca2+-release in remodeled tubules. This work was funded by the European Union 7th Framework Program (FP7/2007- 2013) under grant agreement n° 284464, 241526, by the Italian Ministry of University and Research (NANOMAX), and by Telethon-Italy (GGP13162).
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