Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes
Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes
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DOI：
10.1074/jbc.m115.650531
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发表时间：
2015-08-14
影响因子：
4.8
通讯作者：
Chen, S. R. Wayne
中科院分区：
文献类型：
--
作者：
Hiess, Florian;Vallmitjana, Alexander;Chen, S. R. Wayne
心脏Ca 2+释放通道（Ryanodine receptor，RyR 2）在心肌细胞兴奋-收缩偶联中起重要作用。有效和稳定的兴奋-收缩偶联不仅取决于RyR 2的表达，而且取决于其分布。尽管RyR 2很重要，但人们对RyR 2在活细胞中的分布和组织知之甚少。为了研究RyR 2在活心肌细胞中的分布，我们产生了表达GFP标记的RyR 2（GFP-RyR 2）的敲入小鼠模型。从GFP-RyR 2小鼠心脏分离的活心室肌细胞的共聚焦成像显示GFP-RyR 2簇以条纹图案成行组织。在固定的心室肌细胞中观察到GFP-RyR 2簇的类似组织。用抗α-辅肌动蛋白抗体（Z线标记物）的免疫荧光染色显示，几乎所有GFP-RyR 2簇都定位在Z线区域中。存在具有错位的GFP-RyR 2簇的小区域。有趣的是，这些相同的区域也显示出错位的z线。di-8-ANEPPS染色显示，几乎所有GFP-RyR 2簇均与横管共定位，但与纵管不共定位，而MitoTracker Red染色显示，GFP-RyR 2簇未与活心室肌细胞中的线粒体共定位。我们还发现GFP-RyR 2簇仅在活心室肌细胞的外周处散布在z线之间。GFP-RyR 2簇和Ca 2+火花的同时检测表明，Ca 2+火花仅起源于RyR 2簇。RyR 2簇的Ca 2+火花诱导线粒体Ca 2+水平没有检测到的变化。这些结果首次揭示了RyR 2簇在活心室肌细胞中的分布及其功能相关性。
The cardiac Ca2+ release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-alpha-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca2+ sparks showed that Ca2+ sparks originated exclusively from RyR2 clusters. Ca2+ sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca2+ level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.