F0). Half-maximal Ca2+ release (F50) of the averaged [Ca2+]i transient served as reference and threshold for categorizing local Ca2+ release areas into early and late release areas, respectively near-TTs and more remote. This correlation with the TT network was established earlier [12], and confirmed in the same animal population used in the present study [18]. Early release areas are those that reached F50 within 20 ms after the start of the [Ca2+]i transient; delayed release areas are those that did not. Spontaneous Ca2+ sparks were detected automatically, based on [20] (Cri 4.5). Sparks were assigned to bins of 3 mm width across the scan line. Each bin was linked to the local release area in the averaged [Ca2+]i transient in the corresponding part of the scan line and thus sparks were categorized as occurring near TTs (early release areas) or more remote. Spark properties were subsequently analyzed per category, i.e. associated with early or delayed release sites; spark frequency was likewise normalized within each category, i.e. number of events per area length with early or delayed release sites. Spark width and duration are shown as Full- Width at Half-Maximum (FWHM) and Full-Duration at Half- Maximum (FDHM) respectively. Sparks were considered as extremely long when more than three standard deviations above the average spark duration in CTRL (47.6 ms). Statistics Data are shown as mean 6 SEM, averaged by animal. Comparisons were done using Student’s t-test, or Mann Whitney test, as appropriate. P,0.05 was considered significant. Results Heterogeneous spark properties in pig ventricular myocytes Spontaneously occurring Ca2+ sparks were recorded after stimulation at 1 Hz to load the SR to steady state and assigned to early or delayed release areas according to their position on the scan line, as shown in Fig. 1A. In CTRL myocytes, spark frequency was significantly higher in early than in delayed release areas (Fig. 1B). Spark amplitude and width were comparable (Fig. 1B). Spark duration was significantly shorter in early than in delayed release areas (Fig. 1B). The role of sarcolemmal fluxes in spark frequency and duration Since NCX is known to be highly expressed in TTs [2,21], we investigated whether NCX affects spark duration preferentially in early release areas. Therefore we compared spark duration with and without NCX block (Ni2+, 5 mmol/L NiCl2) applied for the duration of the spark recording, as shown in the inset of Fig. 2. Spark duration was significantly prolonged with Ni2+ in early, but not delayed release areas (Fig. 2A). Yet block of NCX did not alter spark frequency, nor baseline fluorescence (data not shown). Spark amplitude and width were also unchanged (data not shown). As Ni2+ also blocks Ca2+ entry through LTCC, the data suggest Ca2+ entry through LTCC does not influence spark frequency. This was further examined more specifically by comparing sparks with and without LTCC block by Cd2+ (50 mM CdCl2). ICaL inhibition indeed did not affect spark frequency in early or delayed release areas (Fig. 2B). Spark morphology was also unchanged (data not shown). The effect of remodeling in the area adjacent to MI on Ca2+ sparks We first examined the properties of the total of Ca2+ sparks as is usually reported, i.e. without regional analysis. Average spark T-Tubule Proximity Modulates RyR Properties frequency in MI myocytes was not different from CTRL but spark amplitude and width were significantly increased in MI; average spark duration was not different (Fig. 3B). Spark mass, calculated as spark amplitude*width*duration, was significantly higher in MI than in CTRL and the spark-mediated Ca2+ leak from the SR, defined as spark frequency*spark mass, was thus also increased significantly in MI, by nearly three-fold (Fig. 3C). These data must be viewed against the SR Ca2+ content which is a main modulator of leak [22]. SR Ca2+ content was calculated from caffeine- induced Ca2+ release and the accompanying NCX current [18]. Taking into account relative changes in cell content, SR Ca2+ content was significantly higher in MI (123632 mmoles/L vs. was 5265 mmoles/L in CTRL, P,0.05; Fig. 3D). Differential remodeling of spark properties in MI Subsequently, we compared sparks in early and delayed release areas within the MI group. As reported before, due to the decrease in TT density [18], the number and extent of delayed release areas was larger in MI. The fraction of sparks in early areas was reduced (50.666.3% in MI, Npigs = 7, ncells = 33, vs. 60.463.2% in CTRL, Npigs = 12, ncells = 41, P,0.05). We next examined spark param- eters which in CTRL were different between early and delayed sites. In contrast to CTRL, no relation between spark frequency and the type of release area was found in MI (Fig. 4A). This was rather due to an increase in spark frequency in delayed areas than to changes in early areas. In contrast to CTRL animals, spark duration was not different between early and delayed release areas in MI (Fig. 4B). This was due to prolonged spark duration in early areas in MI but not in delayed areas. We also found extremely long sparks (Fig. 4C), categorized as sparks with a duration three standard deviations above the average spark duration in CTRL. The lower threshold for these long sparks was 47.6 ms, but many were found to be longer than 100 ms. The percentage of cells showing long sparks was significantly higher in MI than in CTRL. Reduced NCX removal at the sarcolemma in MI With spark duration prolonged in early, but not delayed areas in MI, we hypothesized that changes in NCX function in MI were responsible for this selective prolongation of near-TT sparks. We had previously reported that global Ca2+ removal by NCX from myocytes was reduced in MI but this could be related to the reduced surface area with loss of TTs; protein expression levels were unchanged [18]. Now we examined removal per surface area as the inward NCX current density on repolarization (Fig. 5B). This was significantly lower in MI than in CTRL, while [Ca2+]i measured at the same time point was comparable (Fig. 5C). Cell culture as a model for extensive loss of TTs To assess the effect of TT loss on RyR function, we studied myocytes cultured for 48 hours (Fig. 6A). As expected, TT density was decreased significantly but, like in MI, RyR density and distribution remained unchanged (Fig. 6A). The fraction of delayed release areas was increased by 31% in culture (P,0.05). On average spark frequency and morphology were comparable between CULT and CTRL (data not shown). Spark mass and calculated leak from sparks was likewise not different and SR Ca2+ content was unchanged in culture (70612 mmoles/L in CULT, Npigs = 6, ncells = 19, vs. 7669 mmoles/L in CTRL, Npigs = 6, ncells = 16, P = NS). Analysis per region (Fig. 6B) showed that spark duration was not different between early and delayed release areas (32.9611.3 ms in early vs. 22.464.0 ms in delayed, Npigs = 7, ncells = 14, P = NS). Compared to CTRL cells, spark PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25100