Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

Abstract

In cardiac myocytes a complex network of membrane tubules -the transverse-axial tubule system (TATS) -controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca 2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization.Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

Axial tubule junctions control rapid calcium signaling in atria

“…Our observations are supported by previously described dedicated AM isolation and high-resolution imaging workflows (18,40), which led to our finding of preserved centrally located AT structures within TAT networks. Since AMs are generally thinner than VMs, it has been proposed that TTs are not needed for ECC in these cells (8,19).…”

“…Although earlier electron microscopy-based (EM-based) representations of AMs (6), recent confocal data, and computational models (19) suggest that there are few, if any, TT invaginations, experimental studies have increasingly highlighted the presence of atrial TAT networks in several mammalian species, including humans (20)(21)(22). To clarify this apparent contradiction, we used membrane-preserving workflows developed for high-resolution live-cell imaging (9,18) that revealed abundant TAT membrane structures in murine AMs ( Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI88241DS1). We identified intact TAT networks in 100% of the AMs examined (n = 29), thus confirming the presence of preserved membrane structures.…”

“…We hypothesize that AM-specific components of the transverse axial tubule (TAT) system combine molecular functions with rapid Ca 2+ signal activation in a highly localized manner in AMs. Our investigations were made possible by recent methodological advances, which provide high-quality samples for imaging of intact AM membrane structures (9,18). While intracellular TAT structures are generally robust and functional throughout AMs, as described below, it was remarkable to observe large axial tubule (AT) structures connected through a sparse pattern of TTs.…”

“…Mouse cells. AMs and VMs were isolated from adult mouse hearts using collagenase and protocols tuned to preserve subcellular structural integrity, as previously described (9,18). RyR2-S2814A +/+ -knockin mice were provided by Xander H.T.…”

“…For AT structure-function analysis, we measured Ca 2+ transients in AM using fluo-4 and the membrane-intercalculating dye di-8-ANEPPS using protocols established previously (9,18). Transversal line scanning during electrical stimulation revealed robust Ca 2+ rises, originating from AT sites ( Figure 1K).…”