Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density Liesbeth Biesmans1, Niall Macquaide1,2, Frank R. Heinzel1,3, Virginie Bito1, Godfrey L. Smith2, Karin R. 1 Sipido * 1 Laboratory of Experimental Cardiology, University of Leuven, Leuven, Belgium, 2 Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom, 3 Division of Cardiology, Medical University of Graz, Graz, Austria Abstract Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI). Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI. Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F.F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category. Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/ Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (.47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI. Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves. Citation: Biesmans L, Macquaide N, Heinzel FR, Bito V, Smith GL, et al. (2011) Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density. PLoS ONE 6(10): e25100. doi:10.1371/journal.pone.0025100 Editor: Marcello Rota, Brigham & Women’s Hospital - Harvard Medical School, United States of America Received July 28, 2011; Accepted August 26, 2011; Published October 13, 2011 Copyright: 2011 Biesmans et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a fellowship to LB by the ‘‘Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT- Vlaanderen),’’ and by grants from the Belgian Science Policy program (IUAP, P6/31) and the European Union (LSHM-CT-2005-018833, EUGeneHeart) and from the EC FP7/2007-2013 under grant agreement no HEALTH-F2-2009-241526, EUTrigTreat (to K.R.S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Karin.Sipido@med.kuleuven.be Introduction In ventricular cardiac myocytes, Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCC) activates Ca2+ release from ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR). This Ca2+- induced Ca2+ release occurs in local release units or couplons, i.e. clusters of LTCC facing clusters of RyR, which are preferentially located in the T-tubules (TT). The preferential location of couplons in TTs [1,2] make TTs critically important for the synchrony of Ca2+ release. Indeed, the [Ca2+]i transient results from spatiotemporal summation of elementary release events, or Ca2+ sparks [3,4], which, in rat ventricular myocytes, are mainly localized near TTs [5,6]. Sparks can also occur during diastole, representing spontaneous probabilistic activation of a RyR or a RyR cluster [3]. TTs are poorly developed or absent in neonatal myocytes and in cells from the specialized conduction system such as Purkinje cells, as well as in atrial cells. These cells are small and show a radial spread of Ca2+ from the external sarcolemma to the center mediated by a combination of Ca2+ release and diffusion [7,8]. Experimental disruption of TTs in ventricular myocytes by osmotic shock or culture has been shown to reduce the synchrony of Ca2+ release, and depress and slow the Ca2+ transient [9–11]. In pig ventricular myocytes, as in human myocytes, TTs are less developed than in rodents [10,12,13]. In contrast, RyRs are distributed homogeneously and regularly throughout the cells, indicating that there is relatively more non-junctional RyRs than in ventricular myocytes of rodents. Ca2+ release during excitation- contraction coupling is consequently less synchronized and areas PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e25100