Shining New Light on the Structural Determinants of Cardiac Couplon Function: Insights From Ten Years of Nanoscale Microscopy

Abstract

Remodelling of the membranes and protein clustering patterns during the pathogenesis of cardiomyopathies has renewed the interest in spatial visualisation of these structures in cardiomyocytes. Coincidental emergence of single molecule (super-resolution) imaging and tomographic electron microscopy tools in the last decade have led to a number of new observations on the structural features of the couplons, the primary sites of excitation-contraction coupling in the heart. In particular, super-resolution and tomographic electron micrographs have revised and refined the classical views of the nanoscale geometries of couplons, t-tubules and the organisation of the principal calcium handling proteins in both healthy and failing hearts. These methods have also allowed the visualisation of some features which were too small to be detected with conventional microscopy tools. With new analytical capabilities such as single-protein mapping, in situ protein quantification, correlative and live cell imaging we are now observing an unprecedented interest in adapting these research tools across the cardiac biophysical research discipline. In this article, we review the depth of the new insights that have been enabled by these tools toward understanding the structure and function of the cardiac couplon. We outline the major challenges that remain in these experiments and emerging avenues of research which will be enabled by these technologies.

Three-Dimensional and Chemical Mapping of Intracellular Signaling Nanodomains in Health and Disease with Enhanced Expansion Microscopy

“…On the other hand, the purely chemical approach of EExM is likely more accessible to cell biologists who often have limited expertise in instrumentation development and the intricacies of fluorophore photochemistry, as argued previously. 62…”

Nano-scale morphology of cardiomyocyte t-tubule/sarcoplasmic reticulum junctions revealed by ultra-rapid high-pressure freezing and electron tomography

“…The variability in TT-jSR membrane distance observed here does not preclude accommodation of RyR, whose cytoplasmic component is thought to extend around 12 nm beyond the SR membrane. Recent molecular imaging results have shown that the in-plane arrangement of RyR is highly non-uniform, with small and larger clusters of RyR whose topology is susceptible to changes in cellular environment [ [46] , [47] , [48] , [49] , [50] , [51] ]. Our observations are consistent with such a dynamic arrangement, and in regions of the dyad lacking RyR, the two membranes may either be pulled closer together (aided, potentially, by junctophilin), or be less well linked and more separated.…”

Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment

“…The calcium transient data suggest improved coupling of RyRs with LTCC to form functional dyads in T3 treated myocytes, even though the total protein content was largely unchanged. Recent advancements in microscopic nanoimaging using STORM technology that allows visualization of single molecules or clusters of RyR and LTCC proteins at the junctional SR-TT microdomains will advance our understanding of the effects of T3 on the formation of the RyR-LTCC dyads in failing cardiomyocytes (Jayasinghe et al 2018; Shen et al 2019).…”