of delayed Ca2+ release were shown to result from local absence of TTs [12]. This study did not show intrinsic differences of RyR related to TT presence, but analysis was limited to the globally triggered release, which may mask such differences. Ca2+ sparks, reflecting intrinsic activity and properties of RyR clusters, may provide more information. In ventricular myocytes these mainly occur at the junctional SR [14] while in atrial cells lacking TTs, sparks occur near the sarcolemma but also in the central areas. Studies in cat atrial cells showed a higher frequency of near-sarcolemmal junctional sparks than of central non- junctional sparks, while amplitude, width and duration were comparable [15], although in rat atrial myocytes the time course of subsarcolemmal sparks was shorter and with a reduced local spread [16]. Data informing whether in ventricular myocytes a sparse TT network could lead to inhomogeneities of RyR cluster properties are limited. In ventricular myocytes from dogs with heart failure, 2D imaging showed non-uniform distribution of sparks related to loss of TTs with less activity in areas devoid of TTs [17]. In the present study, we build on our earlier data where we identified areas without TTs within pig ventricular myocytes based on the local Ca2+ transient properties during normally triggered SR Ca2+ release [12,18]. We use this approach as a tool to assess the effects of TT proximity on spontaneous spark and RyR properties during diastole. We further examine RyR properties in a pig model of ischemic cardiomyopathy, where TT density was significantly reduced [18]. The data show the presence of significant heterogeneity of sparks at baseline with further modulation after remodeling in the area adjacent to a myocardial infarction (MI). Materials and Methods Pig model of ischemic cardiomyopathy Animals were housed and treated according to the Guide for the Care and Use of Laboratory Animals (National Institute of Health, U.S.A.) and experimental protocols were approved by the in-house ethical committee (Ethische Commissie Dierproeven, Katholieke Uni- versiteit Leuven), with permit numbers P02042, P06048 and P10139. The MI data in the current study are derived from the same pigs described earlier [18]. All interventions were performed under full anesthesia (premedication with tiletamine and zolazepam, 4 mg/kg IM and xylazine 0.25 mg/kg IM, maintenance anesthesia with propofol IV, 7 mg/kg/hour; additional analgesia with buprenor- phine HCl 0.1 mg/kg). Implantation of a copper-coated stent in the proximal circumflex coronary artery of young adult domestic pigs (20–25 kg) induced intima proliferation and a high degree stenosis (.90%) within 10–15 days. After 6 weeks, global LV function and extent of MI were evaluated with magnetic resonance imaging; included pigs had only modest reduction of the ejection fraction with an MI that extended on average less than 10% of the LV mass [18]. Pigs were sacrificed 24–48 hrs later (Npigs MI = 7) and compared to healthy weight-matched controls (Npigs CTRL = 12). To evaluate the effect of sarcolemmal fluxes and culture, additional control pigs were used in the present study (Npigs = 12). Isolation of cardiomyocytes Cardiac myocytes were isolated enzymatically as described previously [18]. Briefly, the coronary artery was cannulated distal to the stenosis and the tissue wedge perfused on a Langendorf setup at 37uC. After perfusion with low Ca2+ Tyrode (in mmol/L: NaCl 130, KCl 5.4, KH2PO4 1.2, MgSO4 1.2, CaCl2 0.18, Na- Hepes 6, glucose 20, pH 7.2), the tissue was perfused with 1.4 g/ T-Tubule Proximity Modulates RyR Properties L, collagenase A (Roche, Germany) and 0.1 g/L,protease XIV, (Sigma-Aldrich, Belgium), followed by washout with low Ca2+ tyrode. After removal of the tissue wedge from the setup, the infarct area and border zone were discarded and cells were obtained by gentle trituration of the digested midmyocardial tissue adjacent to the MI. The obtained cell suspension was filtered and resuspended in normal Tyrode (in mmol/L, NaCl 137, KCl 5.4, MgCl2 0.5, CaCl2 1.8, Na-HEPES 11.8, and glucose 10; pH 7.4). CTRL cells were isolated from the same area of matched healthy pigs. In a number of experiments, freshly isolated cardiomyocytes from CTRL pigs were put in primary culture for 48 h as described previously [10]. Cultured cells were compared with freshly isolated cells from the same pigs (Npigs CTRL = 7). Immunofluorescence Myocytes were fixed with 2% paraformaldehyde and permeabi- lised with 0.5% Triton X-100 in PBS. They were washed 3 times and incubated overnight with IgG-anti-RyR primary antibody (clone 34C, 1/100 in PBS, Affinity BioReagents, Golden, CO, USA) at 4uC. The primary antibody was rinsed with PBS and the secondary antibody, FITC-conjugated anti-mouse IgG, was added to the fixed myocytes for 2 hours (1/250 in PBS, Sigma-Aldrich, Belgium). Electrophysiology and global [Ca2+]i measurements Transmembrane ionic currents were recorded using an Axon 200B amplifier using whole-cell voltage clamp in the ruptured patch configuration. Pipette solution contained (in mmol/L: K- aspartate 120, NaCl 10, KCl 20, K-HEPES 10, MgATP 5, and K5fluo-3 0.05; pH 7.2). Inclusion of fluo-3 salt in the pipette solution allowed simultaneous measurements of ionic currents and [Ca2+]i transients. Patch pipettes (GB 200-8P, Science products) had a resistance of 2.0–3.0 MV. All experiments were carried out at 37uC in normal Tyrode solution. Where indicated NiCl2 (5 mmol/L) or CdCl2 (50 mmol/L) were added to this solution and applied through a fast perfusion system. Experiments with Ni2+ were performed in the presence of 1 mmol/L forskolin. [Ca2+]i transients were elicited by depolarizing pulses (250 ms) from 270 to +10 mV at 1 Hz. NCX current density was measured as the inward tail current 10 ms after repolarization. SR Ca2+ content was measured by integrating the inward Na+/ Ca2+ exchange (NCX) current during fast caffeine application (10 mmol/L) and expressed in terms of accessible cell volume. Confocal microscopy Spatiotemporal characteristics of [Ca2+]i transients and sponta- neous Ca2+ sparks were studied using a Zeiss Axiovert 100 M inverted microscope with a 640/1.3 oil-immersion objective in combination with a Zeiss LSM 510 confocal laser point-scanning system (LSM 510, Zeiss, Jena, Germany). Quiescent, rod-shaped myocytes with clear striations were randomly selected. The 488 nm line of a 25 mW argon laser was used for excitation of Fluo-3. Ca2+ release was visualized by repetitive scanning of a line selected longitudinally through the cell, avoiding the nuclei. X and Y resolution were 0.2 to 0.4 mm and 1.54 ms respectively. Stimulated Ca2+ transients were recorded after reaching a steady state. Stimulation was subsequently stopped and spontaneous Ca2+ sparks were recorded for 15 s. Image analysis Linescan images of [Ca2+]i transients and spontaneous Ca2+ sparks were analyzed using a custom-made program, modified after [19]. Fluorescence was normalized to the diastolic [Ca2+]i level (F/ PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e25100