Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes.

Methods and Results— Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈40% greater current. Optical mapping of Ca 2+ transients revealed that rat AMs presented ≈3-fold as many spontaneous Ca 2+ release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca 2+ transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-β-cyclodextrin, with an associated ≈30% whole-cell I Ca,L reduction. Heart failure (16 weeks post–myocardial infarction) in rats resulted in a T-tubule degradation (by ≈40%) and significant elevation of spontaneous Ca 2+ release events. Although heart failure did not affect LTCC occurrence, it led to ≈25% decrease in T-tubule LTCC amplitude.

Materials and Methods

Detailed methods are provided in the online-only Data Supplement Methods. All studies complied with the United Kingdom Home Office regulation governing the care and use of laboratory animals and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Myocyte Isolation, T-Tubule Characterization, and Animal Models Single atrial myocytes were isolated separately from both left (LA) and right (RA) atrial of control and 16-week post–myocardial infarction rats (online-only Data Supplement Table I). The subcellular T-tubule system was visualized by confocal imaging of Di-8-ANEPPS–stained cells. Surface topography was characterized by scanning ion conductance microscopy which uses a glass nanopipette as a sensitive probe. 19 Super-resolution Scanning Patch-Clamp With Pipette Clipping Modification After generating a topographical image of the cell surface by scanning ion conductance microscopy, the tip diameter of the pipette was widened by clipping 19 to increase the area of attachment. The pipette was then lowered to a specific location until it touched the membrane, and a high-resistance seal was established. Recordings were then performed in a cell-attached mode. Controlled widening of the scanning nanopipette tip is described in detail in the online-only Data Supplement Methods. Macroscopic calcium currents were recorded by using the whole-cell patch-clamp technique. 20 Optical Mapping and Data Analysis Optical mapping of cells loaded with the Ca 2+ -sensitive fluorescent dye Fluo-4 AM via a complementary metal-oxide semiconductor camera ULTIMA-L (SciMedia, USA Ltd, Costa Mesa, CA) was used to monitor localized changes in [Ca 2+ ] i .

Statistical Analysis

All graphs and statistical analysis were performed by using either GraphPad prism 5 or Origin version 6.1. The average values were calculated throughout all cells studied within the groups and then compared between the groups. Normality was tested using the Kolmogorov-Smirnov test. In cases where data failed the normality test, the nonparametric Mann-Whitney test was used instead of the unpaired Student t test, and the nonparametric Kruskal-Wallis test was used instead of analysis of variance. Statistical differences were assessed with analysis of variance, Student t test, Mann-Whitney test, Kruskal-Wallis test, χ 2 , and Fisher exact test as appropriate. All data are expressed as mean±standard error of the mean. A value of P