Analysis of Cav1.2 and Ryanodine Receptor Clusters in Rat Ventricular Myocytes

Abstract

We analyzed the distribution of ryanodine receptor (RyR) and Cav1.2 clusters in adult rat ventricular myocytes using three-dimensional object-based colocalization metrics. We found that ∼75% of the Cav1.2 clusters and 65% of the RyR clusters were within couplons, and both were roughly two and a half times larger than their extradyadic counterparts. Within a couplon, Cav1.2 was concentrated near the center of the underlying RyR cluster and accounted for ∼67% of its size. These data, together with previous findings from binding studies, enable us to estimate that a couplon contains 74 RyR tetramers and 10 copies of the α-subunit of Cav1.2. Extradyadic clusters of RyR contained ∼30 tetramers, whereas the extradyadic Cav1.2 clusters contained, on average, only four channels. Between 80% and 85% of both RyR and Cav1.2 molecules are within couplons. RyR clusters were in the closest proximity, with a median nearest-neighbor distance of 552 nm; comparable values for Cav1.2 clusters and couplons were 619 nm and 735 nm, respectively. Extradyadic RyR clusters were significantly closer together (624 nm) and closer to the couplons (674 nm) than the couplons were to each other. In contrast, the extradyadic clusters of Cav1.2 showed no preferential localization and were broadly distributed. These results provide a wealth of morphometric data that are essential for understanding intracellular Ca2+ regulation and modeling Ca2+ dynamics.

Upregulation of the kappa opioidergic system in left ventricular rat myocardium in response to volume overload

“…To confirm the findings of our binding studies we performed double immunofluorescence confocal microscopy in which KOR colocalized with the sarcolemma and invaginated T-tubules bound calcium channel Ca v 1.2 located in left ventricular cardiomyocytes of control rats [32]. KOR also colocalized with the intracellular calcium channel RyR of the sarcoplasmatic reticulum.…”

Nanoscale analysis of ryanodine receptor clusters in dyadic couplings of rat cardiac myocytes

“…1 A). Staining was characteristically absent in spaces presumably occupied by myofibrils, mitochondria and nuclei, a labelling pattern qualitatively similar to diffraction-limited transverse images of RyR immunofluorescence staining in rat ventricular myocytes in previous studies [6,8,29]. Upon close examination of super-resolution images, we observed a wide range of sizes of RyR clusters some of which exhibited highly elongated or other, more complex shapes (Fig.…”

Junctional Cleft [Ca 2+ ] i Measurements Using Novel Cleft-Targeted Ca 2+ Sensors

“…38 Thus, the GCaMP2.2-FKBP12.6 signal is not generated entirely by [Ca 2+ ] Cleft , but is slightly contaminated by [Ca 2+ ] at such non-junctional RyRs. Of note, local [Ca 2+ ] at non-junctional RyRs may also be relevant, even if less influential on sarcolemmal currents.…”