In sheep atrial myocytes, a moderate density of t-tubular structure has been described, which was further reduced as a result of remodeling in a model of atrial pacing induced persistent atrial fibrillation (AF). A concomitant reduction in ICaL and triggered Ca2+ release was observed, as well as attenuation of β-receptor responsiveness. In the current study, we have examined the effect of proximity of TTs to RyR on diastolic RyR activity (spontaneous Ca2+ sparks) and how this changes after induction of AF. The role of CaMKII in this situation is further investigated. Ca2+ transients were recorded in fluo-4 loaded cells using confocal linescan microscopy (CLSM). Whole cell voltage clamp was used to elicit Ca2+ release induced by depolarizing pulses at 2Hz. 3-D distance mapping was used to correlate time to half maximal release of the fluorescence from triggered Ca2+ release with TT proximity. Release sites were localized and categorized as coupled (≤0.5 µm) and non-coupled (≥2 µm). After 2 minutes, stimulation was stopped and spontaneous Ca2+ sparks were measured for a period of 15 seconds and assigned to their cellular locations. SR content was assessed by integrating the sodium calcium exchange current after rapid application of caffeine (10 mM). In CTRL myocytes (N=6, n=21), non-coupled sites regions had less frequent sparks (-32.7±11.1%) than coupled regions. These had similar amplitude and width, but the mean half-maximal duration was reduced by 16.5±7.8 %. After AF no change in SR content was observed (N=6, n=23). The most striking difference was observed as a reversal of the regional frequency relationship: sparks were more frequent in non-coupled (+99±16%) vs. coupled regions in atrial myocytes from AF animals. Addition of AIP (5µM) caused a normalization in both AF and CTRL, with no change in SR content. To further activate CaMKII, but in the absence of PKA activation, isoproterenol (100 nM) and PKI (5 µM) was added. This led to the initiation of Ca2+ waves in 50% of CTRL cells and 93% of AF cells.