T-Tubule Proximity Modulates RyR Properties Figure 5. NCX in myocytes from the area adjacent to MI. A. NCX function was measured as the tail current upon repolarization to 270 mV (arrow) after a step to +10 mV in CTRL and MI. B. Averaged NCX current density in CTRL (Npigs = 9, ncells = 30) and MI (Npigs = 7, ncells = 19), with [Ca2+]i measured simultaneously. * denotes P,0.05. doi:10.1371/journal.pone.0025100.g005 However, the observation that spark duration was similar in all areas after Ni2+ argues against this hypothesis. The mechanism of modulation of spark duration by NCX is at present not resolved. A straightforward interpretation is a direct modulation of local Ca2+ by NCX as Ca2+ removal system. Trafford et al. [33] showed that spontaneous Ca2+ waves and caffeine-elicited NCX currents had two components, of which they attributed the first part to near-sarcolemmal, dyadic, release which rapidly and locally activated NCX. Recent data from our group on [Ca2+] near release sites indicates that a fraction of NCX indeed senses high local [Ca2+] during triggered SR Ca2+ release and can thus contribute significantly to local Ca2+ removal [34]. Similarly, NCX can be activated locally by junctional Ca2+ sparks and thus reduce spark duration specifically in areas close to TTs. In contrast, some previous studies have shown that (global) spark duration was not affected by NCX inhibition in rat ventricular myocytes [32,35]. This effect might be species-dependent, with a relatively low dependence of Ca2+ removal on NCX in rat compared to larger mammals [36]. An alternative explanation is that the local removal of Ca2+ by NCX reduces the duration of RyR openings. Modulation of RyR gating by NCX has been studied mainly with regard to the probability of opening. Several studies support the idea that reverse mode NCX during depolarization provides an additional trigger promoting RyR opening [37,38]. In the current study where sparks occur at resting membrane potential, NCX will predominantly act in forward mode. If NCX acts through an effect on gating, one could expect an additional effect of NCX block on frequency of sparks. There was indeed a trend to higher frequency, but this was not significant. Taken together, NCX modulates spark duration near TTs most likely directly through increased removal of local Ca2+ with or without an additional indirect modulation of RyR gating. Effect of cellular remodeling in the area adjacent to MI on near-TT and non-coupled sparks Global spark mass and calculated spark-mediated leak were increased significantly in MI. Whereas at first this feature seems to compare to observations in non-ischemic heart failure models [39,40] or the changes in the rat with heart failure after MI [41], further analysis shows several differences. Unlike in heart failure models, the global increase in spark mass was not primarily mediated through an increase in average spark frequency. Increase in spark mass was primarily due to a higher average amount of Ca2+ released per spark with larger width and amplitude. These data are consistent with the increased SR Ca2+ content, a feature not observed in heart failure. The relation between an increase of SR Ca2+ content and increased spark size [42] or total SR Ca2+ leak [22] is well-established. For a similar increase in SR Ca2+ content, leak measured as increase in cytosolic [Ca2+] in normal rabbit myocytes, increased two-threefold [22], i.e. in the same range as the present data. While this analysis suggests that the increase in SR content in MI can account for the higher leak, more explicit measurements of leak are needed. Indeed, recent data suggest that a substantial leak can occur without visible sparks [43,44]. Further subcellular analysis shows heterogeneity in the changes in local spark properties. Such heterogeneity in local Ca2+ release was also described in the dog with pacing-induced heart failure [17]. In this model, the loss of TTs led to areas with reduced PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e25100