Cardiac cell ultrastructure under the confocal microscope

Fig. 1: A cross-sectional view of a rat ventricular myocyte showing the dense network of t-tubules spanning through the interior of the cell (shown in red, visualized using t-tubular marker Caveolin-3) and clusters of ryanodine receptors (shown in green).

Forceful contraction of heart muscle is crucial in ensuring adequate pumping of blood throughout the circulation. This is achieved by the rapid conversion of action potentials that propagate along the plasma membranes of myocytes into steep and synchronous release of calcium (Ca²⁺) into the myoplasm from the sarcoplasmic reticulum, which is closely followed by the uniform activation of contraction. Invaginations of the plasma membrane forming the transverse tubular network (t-system) are responsible for the synchrony in the Ca²⁺ release at the junctions where the Ca²⁺ release channels (ryanodine receptors) reside in quasi-cystalline clusters. Their opening is triggered by Ca²⁺ ions locally introduced by voltage dependent L-type Ca²⁺ channels present in near vicinity. Another potential source of such trigger Ca²⁺ is the Na⁺/Ca²⁺ exchanger which can mediate Ca²⁺ influx in the early phases of the action potential although the placement of the exchanger (and therefore its effectiveness in triggering release) is unclear due to lack of resolution in conventional confocal data published previously.

Visualising the t-system in rat ventricular myocytes

Using a novel protocol of confocal microscopy combined with fluorescent immunocytochemistry, we have visualized the t-system in rat ventricular myocytes and the localization of ryanodine receptor clusters in relation to the complex network of t-tubules (See 3D PDF, calibration box: 1 micron; in Organization of ryanodine receptors, transverse tubules, and sodium-calcium exchanger in rat myocytes. This data allowed the detection of a sub population of Ca²⁺release sites that were apparently non-junctional and were unlikely to be triggered directly by Ca²⁺ entering via L-type Ca²⁺ channels. Improved co-localization analyses were developed for analyzing similar data of the distribution of the Na⁺/Ca²⁺ exchanger which confirmed the junctional localization of a small fraction of the exchanger proteins.

Revealing sub clusters

More recently, our work has detailed the fine structure of the ryanodine receptor clusters found within junctions near the cell surface. Applying a single molecule localization microscopy technique, enzymatically isolated and fixed ventricular myocytes labeled with commonly used fluorophores linked to secondary antibodies, the localization of ryanodine receptors were reconstructed at ~30-nm resolution. This data revealed that ryanodine receptor clusters seen in confocal and conventional fluorescence micrographs consist of sub clusters that may open in concert. Resolved clusters had complex shapes and size distributions (See attached Figure 2. Published article: Baddeley et al 2009).

Making detailed spatial measurements

One of the principal aims of this work is to make detailed spatial measurements that would help further our understanding of the excitation-contraction coupling in healthy and failing cardiac muscle. The complexity of the sub-cellular structures observed so far have underscored importance in the organization of these protein complexes in the nanometer scale where much of the signaling takes place.