Three-dimensional electron microscopy reveals new details of membrane systems for Ca2+ signaling in the heart

Abstract

In the current study, the three-dimensional (3D) topologies of dyadic clefts and associated membrane organelles were mapped in mouse ventricular myocardium using electron tomography. The morphological details and the distribution of membrane systems, including transverse tubules (T-tubules), junctional sarcoplasmic reticulum (SR) and vicinal mitochondria, were determined and presumed to be crucial for controlling cardiac Ca2+ dynamics. The geometric complexity of T-tubules that varied in diameter with frequent branching was clarified. Dyadic clefts were intricately shaped and remarkably small (average 4.39×105 nm3, median 2.81×105 nm3). Although a dyadic cleft of average size could hold maximum 43 ryanodine receptor (RyR) tetramers, more than one-third of clefts were smaller than the size that is able to package as many as 15 RyR tetramers. The dyadic clefts were also adjacent to one another (average end-to-end distance to the nearest dyadic cleft, 19.9 nm) and were distributed irregularly along T-tubule branches. Electron-dense structures that linked membrane organelles were frequently observed between mitochondrial outer membranes and SR or T-tubules. We, thus, propose that the topology of dyadic clefts and the neighboring cellular micro-architecture are the major determinants of the local control of Ca2+ in the heart, including the establishment of the quantal nature of SR Ca2+ releases (e.g. Ca2+ sparks).

Optical single-channel resolution imaging of the ryanodine receptor distribution in rat cardiac myocytes

“…2D shows that the majority of RyRs are found in somewhat larger clusters of Ϸ25 RyRs. Some clusters were incompletely filled with RyRs, an observation consistent with a recent electron microscopic study which has shown interior junctions to be incompletely filled with RyRs (24).…”

Size Matters: Ryanodine Receptor Cluster Size Affects Arrhythmogenic Sarcoplasmic Reticulum Calcium Release

“…First we assume that our mean cluster ΔF/F 0 of 1.39 (Figure 1D) corresponds to the average (or most frequent) RyR cluster size estimated from several prior studies that range mostly between 50 and 150 RyR/cluster, which in some high‐resolution cases were clusters of clusters or superclusters 2, 3, 4, 5, 6, 27. The mean ΔF/F 0 of 1.39 is indicated by a vertical broken line in Figure 1E.…”

“…Confocal resolution used here cannot directly resolve individual RyRs within a junctional cluster as is possible with superresolution or electron microscopy 2, 3, 4, 5, 6. However, the relative F‐FKBP fluorescence intensity (ΔF/F 0 ) within 250 nm around the local cluster peak intensity (Figure 1D) provides a direct quantitative readout that is proportional to the number of RyRs within that local cluster (or supercluster; Figure 1E).…”

“…High‐resolution imaging has shown that RyR clusters can range from 1 to 400 per cluster,2, 3, 4, 5, 6 but terminology merits further discussion. Seminal quantitative electron microscopy by Franzini‐Armstrong et al4 inferred a typical RyR cluster in rat ventricular myocyte junctions to be 267 in circular arrays, requiring a fully packed checkerboard array of 553 nm diameter (for 30‐nm center‐center RyR distance) 41, 42.…”

“…RyR sensitivity to cytosolic Ca 2+ ([Ca 2+ ] i ) is increased in genetic diseases (catecholaminergic polymorphic ventricular tachycardia), on increased SR Ca 2+ content, during heart failure, and on certain posttranslational modifications (eg, phosphorylation or oxidation), all of which promote diastolic SR Ca 2+ leak and triggered arrhythmias 1. In cardiac myocytes, RyRs form clusters in which some RyRs are tightly packed in checkerboard arrays (corner to corner) with others clustered in a less organized manner within junctional clefts between the SR and sarcolemma 2, 3, 4, 5, 6. These junctional clefts are cylinders 100 to 200 nm in diameter along transverse tubules and surface sarcolemma, with a depth comparable to the height of the RyR protruding from the SR (≈13 nm).…”