T-Tubule Proximity Modulates RyR Properties Figure 6. Modulation of Ca2+ sparks with TT loss during culture. A. Representative confocal images of TT staining with di-8-ANEPPS (left) and RyR staining (right) after 48 hours of culture. B. Representative line scan image of Ca2+ spark protocol in CULT. The type of release areas is marked in blue and red for early and delayed release areas respectively. C. Comparison of spark duration in early (left) and delayed (right) release areas in CTRL (Npigs = 7, ncells = 29) vs. CULT (Npigs = 7, ncells = 14). D. Fraction of cells showing long-lasting sparks (.47.6 ms) in CTRL (17/35 cells with long sparks) and CULT (10/15 cells with long sparks). E. NCX current density in CTRL (Npigs = 4, ncells = 14) and CULT (Npigs = 4, ncells = 7). * denotes P,0.05. doi:10.1371/journal.pone.0025100.g006 sparks and a larger beat-to-beat spark variance. In the present data, an increase in frequency was seen in delayed areas, though not in early areas. The former is consistent with the expectation of increased frequency related to a higher SR Ca2+ content. The latter, the absence of increase in early release sites, could be related to changes in the dyadic environment, such as a reduction in the amount of L-type Ca2+ channels per couplon [18], a change in the dyadic space geometry, or a reduction in RyR Po, which may offset the effect of an increased SR Ca2+ content. This needs further investigation. Spark duration increased preferentially near TTs in MI, suggesting that local NCX Ca2+ removal is reduced. The reduced NCX current density is consistent with this hypothesis, which is further supported by analysis of the phases of Ca2+ removal during caffeine application (Fig. 7) [33]. In the majority of CTRL cells (62%), two clear phases of NCX mediated efflux were evident (Fig.7A). In MI only 40% showed this type of release, with a trend to a longer Taufast (Fig.7B), indicating a smaller component of NCX Ca2+ removal from junctional release sites, and thus a reduction of T-tubular NCX. These results suggest a redistribution of NCX transporters between the TT and other sarcolemmal compartments, since global NCX expression remained unchanged after MI [18]. Interestingly, an increase in the amount of cells with long sparks was observed in MI. Since the occurrence of long sparks was not different between the types of release areas, the mechanism for long sparks is probably different from the NCX-mediated regulation of spark duration described above. Long sparks are most likely related to altered gating properties of RyR clusters. These have been reported earlier to correlate with RyR subconductance states [45] and with downregulation of FK506- binding proteins FKBP12 and FKBP12.6 [46]. Computational PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e25100