Research Highlight

Three Types of Microscopy Go To the Limit to Determine Ryanodine Receptor Calcium Release Channels in Cardiac Myocytes



Figure Caption: Views of caveolae and sub-sarcolemmal (SR) at the surface sarcolemma. This figure shows several views of sarcolemma (blue), surface caveolae (green), couplings (red) and sub-sarcolemmal SR (pale yellow) A. View of the sarcolemma from the cytoplasmic side. Panels B-D look onto the sarcolemma slightly from the extracellular side.  Caveolae openings are visible in green in B (see arrows). The inset in C shows a “thin section micrograph” extracted as a slice from the tomogram in which electron-dense “feet” are visible spanning the gap between caveolar (green) and peripheral SR (pale yellow) membranes, see arrows. The anatomical features were segmented from an EM tomogram data set shown in this paper. Scale bars, A 200 nm; B-D 100 nm; C inset 50nm.

January, 2014 La Jolla -- A research team led by UCSD recently published results of their work using various kinds of microscopy to reveal the relative distribution of ryanodine receptors (RyRs) and caveolin-3 (CAV3) in mouse ventricular myocytes in the cytosol and near the cell surface. RyRs are important to investigate because they comprise a class of intracellular calcium channels that mediate the release of calcium in various types of mammalian tissue like muscles and neurons. The contraction of cardiac ventricular myocytes depends on the rapid, cell-wide Ca2+ transient increase when the cell-membrane potential is depolarized. The cardiac RyR, which is the intracellular Ca2+ release channel in the sarcoplasmic reticulum (SR), plays a central role in shaping these transients.

NCMIR scientists and Dr. Masahiko Hoshijima in the Department of Medicine in collaboration with Dr. Soeller’s group in New Zealand used super-resolution fluorescence light microscopy (LM) to achieve a resolution approaching 30nm. They also used 3D electron microscopy (EM) and serial block-face scanning EM (SBEM), which provided even higher resolution (down to smaller than 10nm). To their knowledge, it was the first time EM had been applied to study mouse ventricular tissue. All three systems were provided by the National Center for Microscopy and Imaging Research, led by Mark H. Ellisman at UCSD. The data resulting from this work demonstrate that complementary data from optical super-resolution and 3D EM images ensures data integrity across scales and supports data interpretation.

RyRs form clusters of various sizes with the majority located within junctions between the SR and the surface membrane and its cytoplasmic extension, the transverse tubular (t-) system. Some RyR clusters are believed to be associated with caveolae, a specialized signaling microdomain of the surface membrane.

Previous studies were complicated by the limited resolution of optical imaging methods of ~250nm, much larger than the nanometer scale of RyRs and caveolae. As a result, these studies report varying colocalization between RyRs and CAV3, a caveolar marker also expressed in the t-system.

The images of RyR and CAV3 labeling at the surface sarcolemma of mouse myocytes showed little overlap, suggesting that few RyRs were in couplings with caveolae. More specifically, quantitative analysis of data at the surface sarcolemma showed that 4.8% of RyR labeling colocalized with CAV3, whereas 3.5% of CAV3 was in areas with RyR labeling. The association of the two labels increased slightly several microns below the surface sarcolemma. Values corresponding to those above increased to 9.2 and 9.0%, respectively, in the interior of myocytes where CAV3 was widely expressed in the t-system but reduced in regions associated with junctional couplings. EM tomography independently showed only a few couplings with caveolae and little evidence for caveolar shapes on the t-system.

The team suggests that this regional specialization helps reduce ionic accumulation and depletion in transverse tubule (T-tubule) system lumen during excitation-contraction coupling to ensure effective local Ca2+ release.

Unexpectedly, super-resolution LM and 3D EM data both revealed significant increases in local t-system diameters in many regions of strong RYR labeling. This conclusion was supported by two different 3D EM modalities with less than 10nm resolution: EM tomography and SBEM. The latter is related volume EM technique to study larger cells volumes but at the expense of slightly lower resolution.

More fundamentally, this data demonstrate that super-resolution LM and volume EM techniques complementarily enhance information on subcellular structure at the nanoscale. Correlating optical and EM imaging approaches in this way should provide further information and continue to improve our knowledge of the basis of cardiac excitation-contraction coupling.

In addition to UCSD, the team was comprised of researchers from the University of Auckland (New Zealand), University of Wisconsin-Milwaukee, Yale University, and the University of Exeter (UK).

Citation: Relevant publication: Wong, Joseph, David Baddeley, Eric A. Bushong, Zeyun Yu, Mark H. Ellisman, Masahiko Hoshijima, and Christian Soeller, Nanoscale Distribution of Ryanodine Receptors and Caveolin-3 in Mouse Ventricular Myocytes: Dilation of TTubules Near Junctions, Biophysical Journal, 2013, 104: L22-L24,  

Link to Online Article

Supporting imagery and two movies