T-Tubule Proximity Modulates RyR Properties Figure 4. Subcellular spark properties in myocytes from the area adjacent to MI. A. Spark frequency, and, B, duration in early vs. delayed release areas in MI, with the MI-dependent change in each area (CTRL Npigs = 12, ncells = 41 vs. MI Npigs = 7, ncells = 33). C. Example of long spark in 3D and fraction of cells showing long-lasting sparks (.47.6 ms) in CTRL (24/43 cells with long sparks) and MI (22/31 cells with long sparks). * denotes P,0.05. doi:10.1371/journal.pone.0025100.g004 colocalized with TTs [17], consistent with the results of the present study. In rat atrial myocytes, there is also a higher propensity for spontaneous Ca2+ sparks in the junctional SR [24]. One possible mechanism for a higher spark frequency near TTs is background Ca2+ entry through L-type Ca2+ channels. However, block of ICaL did not affect spark frequency in early release areas. This is in agreement with earlier studies showing that ICaL block or removal of extracellular Ca2+ did not affect spark frequency in rat ventricular myocytes, indicating sparks were indeed ‘spontaneous’ and did not require sarcolemmal Ca2+ influx. In atrial myocytes from the cat, Ca2+ influx was important for the higher spark frequency near the subsarcolemma [15], but in rat atrial myocytes it was not [25]. The latter results indicate that the mere presence of sarcolemma, and LTCC, may play a role. Indeed, presence and activation of LTCC per se can enhance RyR activity even without enhanced Ca2+ influx, as shown by the effects of BayK in rat ventricular myocytes [26]. Further evidence comes from studies on the development of spark activity in fetal and neonatal cardiac cells which are devoid of TTs. Sparks are at first absent but appear once membrane invaginations develop and LTCCs colocalize with RyRs [27]. Initially, this occurs predom- inantly near the sarcolemmal membrane [27,28]. Alternatively, RyRs next to TT membrane may be exposed to elevated local [Ca2+] during excitation-contraction coupling which can lead to activation of CaMKII in the dyad [29,30]. This would result in enhanced activation of RyR near TT. Local differences in SR Ca2+ content are unlikely to underlie the observed differences. Zima et al. [31] showed that not all release sites re-fill at the same rate after sparks, indicating that temporarily gradients may exist, but these are short lived (,100 ms). Further study, using intra-SR low affinity Ca2+ probes is needed to investigate the possible existence of standing gradients in the SR. However, earlier data on caffeine-induced release in different areas suggest that there are no differences in local SR Ca2+ availability on the timeframe of the measurements carried out in the present study [12]. Regulation of spark duration by NCX Detubulation in rat ventricular cells prolonged sparks, indicat- ing a role for sarcolemmal Ca2+ flux in modulation of spark duration [11]. Blocking NCX indeed prolonged sparks specifically in early areas. The shorter duration could also result from a locally higher SERCA activity [32] as a higher local Ca2+ in the dyad could activate SERCA to a larger extent in near-TT sparks. PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e25100