Department of Physiology, University of Auckland, Auckland, New Zealand.
Biomedical Physics, University of Exeter, UK.
J Mol Cell Cardiol. 2015 Mar;80:45-55. doi: 10.1016/j.yjmcc.2014.12.013. Epub 2014 Dec 20.
The contractile properties of cardiac myocytes depend on the calcium (Ca(2+)) released by clusters of ryanodine receptors (RyRs) throughout the myoplasm. Accurate quantification of the spatial distribution of RyRs has previously been challenging due to the comparatively low resolution in optical microscopy. We have combined single-molecule localisation microscopy (SMLM) in a super-resolution modality known as dSTORM with immunofluorescence staining of tissue sections of rat ventricles to resolve a wide, near-exponential size distribution of RyR clusters that lined on average ~57% of the perimeter of each myofibril. The average size of internal couplons is ~63 RyRs (nearly 4 times larger than that of peripheral couplons) and the largest clusters contain many hundreds of RyRs. Similar to previous observations in peripheral couplons, we observe many clusters with one or few receptors; however ≥80% of the total RyRs were detected in clusters containing ≥100 receptors. ~56% of all clusters were within an edge-to-edge distance sufficiently close to co-activate via Ca(2+)-induced Ca(2+) release (100nm) and were grouped into 'superclusters'. The co-location of superclusters with the same or adjacent t-tubular connections in dual-colour super-resolution images suggested that member sub-clusters may be exposed to similar local luminal Ca(2+) levels. Dual-colour dSTORM revealed high co-localisation between the cardiac junctional protein junctophilin-2 (JPH2) and RyR clusters that confirmed that the majority of the RyR clusters observed are dyadic. The increased sensitivity of super-resolution images revealed approximately twice as many RyR clusters (2.2clusters/μm(3)) compared to previous confocal measurements. We show that, in general, the differences of previous confocal estimates are largely attributable to the limited spatial resolution of diffraction-limited imaging. The new data can be used to inform the construction of detailed mechanistic models of cardiac Ca(2+) signalling.
心肌细胞的收缩特性取决于肌浆中兰尼碱受体（RyR）簇释放的钙离子（Ca(2+)）。由于在光学显微镜下的分辨率相对较低，以前准确量化 RyR 的空间分布一直具有挑战性。我们将单分子定位显微镜（SMLM）与大鼠心室组织切片的免疫荧光染色相结合，采用一种称为 dSTORM 的超分辨率模式，以解析 RyR 簇的广泛的、近指数分布。这些 RyR 簇沿肌原纤维的周长平均排列约 57%，平均大小约为 63 个 RyR（几乎是周边 couplons 的 4 倍），最大的簇包含数百个 RyR。与以前在周边 couplons 中的观察结果类似，我们观察到许多具有一个或几个受体的簇；然而，≥80%的 RyR 存在于含有≥100 个受体的簇中。约 56%的所有簇之间的边缘到边缘距离足够近，可以通过 Ca(2+)诱导的 Ca(2+)释放（100nm）协同激活，并被分组为“超级簇”。在双色超分辨率图像中，超级簇与相同或相邻的 t-管连接的共定位表明，成员子簇可能暴露于相似的局部腔 Ca(2+)水平。双色 dSTORM 揭示了心脏连接蛋白 junctophilin-2 (JPH2) 和 RyR 簇之间的高共定位，这证实了观察到的大多数 RyR 簇都是二联体。超分辨率图像的更高灵敏度显示，与以前的共聚焦测量相比，RyR 簇的数量增加了约两倍（2.2 个簇/μm(3))。我们表明，一般来说，以前的共聚焦估计的差异主要归因于衍射受限成像的有限空间分辨率。新数据可用于为心脏 Ca(2+)信号转导的详细机制模型的构建提供信息。
