Clustering of L-Type Cav1.2 Channels in Cardiomyocytes Visualized by a Custom-Built Fast Scanning STED Microscope

Abstract

We directly visualize the cluster size distribution of the L-type Cav1.2 channels in isolated murine cardiomyocytes using super-resolution stimulated emission depletion (STED) microscopy and with antibody labeling. Cav1.2 channels play an essential role in excitation-contraction coupling of cardiac muscle by driving calcium ions into the cardiomyocytes. In fact, it has recently been shown that Cav1.2 channels can form functionally coupled small clusters to amplify the calcium ion influx. Such clustering of the Cav1.2 channels could not be directly observed due to the diffraction-limited resolution of conventional microscopes. We built a resonant scanning STED microscope achieving a lateral resolution of ∼50 nm in a 60μm x 60 μm scanning area acquired at 16 nm per pixel. An 8 KHz resonant scanning mirror provides 16,000 lines per second imaging speed to shorten the exposure time in a single round of scanning, while between consecutive rounds of scanning, fluorophores may relax from the triplet states to the ground states rather than be photobleached. To keep up with the fast imaging speed, the output of the photomultiplier are sampled at 1.8 GHz to grab every detected photon. This microscope enabled us to clearly visualize the clusterized distribution of Cav1.2 channels immunolabeled by fluorescent dyes in isolated murine cardiomyocytes. We show in three different preparations that >80% of the Cav1.2 channels form larger clusters of size 100-150 nm along the T-tubules, and smaller clusters with a typical size of 50 nm (down to the resolution limit of our microscope) are inclined to be present between the T-tubules and on the periphery of the cells. These results provide additional evidence supporting the view that clustering enhances the functional role of Cav1.2 channels. Supported by NIH BRG RO1 HL088640.