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Cardiac myocytes rely on transverse (t)-tubules to facilitate a rapid rise in calcium throughout the cell. However, despite their importance in triggering synchronous Ca 2+ release, t-tubules are highly labile structures. They develop postnatally, increase in density during exercise training and are lost in diseases such as heart failure (HF). In the majority of settings, an absence of t-tubules decreases function. Here we show that despite reduced t-tubule density due to immature t-tubules, the newborn atrium is highly specialised to maintain Ca 2+ release. To compensate for fewer t-tubules triggering a central rise in Ca 2+ , Ca 2+ release at sites on the cell surface is enhanced in the newborn, exceeding that at all Ca 2+ release sites in the adult. Using electron and super resolution microscopy to investigate myocyte ultrastructure, we found that newborn atrial cells had enlarged surface sarcoplasmic reticulum and larger, more closely spaced surface and central ryanodine receptor clusters. We suggest that these adaptations mediate enhanced Ca 2+ release at the sarcolemma and aid propagation to compensate for reduced t-tubule density in the neonatal atrium., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Electron microscopy techniques have made a significant contribution towards understanding muscle physiology since the 1950s. Subsequent advances in hardware and software have led to major breakthroughs in terms of image resolution as well as the ability to generate three-dimensional (3D) data essential for linking structure to function and dysfunction. In this methodological review we consider the application of a relatively new technique, serial block face scanning electron microscopy (SBF-SEM), for the study of cardiac muscle morphology. Employing SBF-SEM we have generated 3D data for cardiac myocytes within the myocardium with a voxel size of ~15 nm in the X-Y plane and 50 nm in the Z-direction. We describe how SBF-SEM can be used in conjunction with selective staining techniques to reveal the 3D cellular organisation and the relationship between the t-tubule (t-t) and sarcoplasmic reticulum (SR) networks. These methods describe how SBF-SEM can be used to provide qualitative data to investigate the organisation of the dyad, a specialised calcium microdomain formed between the t-ts and the junctional portion of the SR (jSR). We further describe how image analysis methods may be applied to interrogate the 3D volumes to provide quantitative data such as the volume of the cell occupied by the t-t and SR membranes and the volumes and surface area of jSR patches. We consider the strengths and weaknesses of the SBF-SEM technique, pitfalls in sample preparation together with tips and methods for image analysis. By providing a 'big picture' view at high resolutions, in comparison to conventional confocal microscopy, SBF-SEM represents a paradigm shift for imaging cellular networks in their native environment., (Copyright © 2014. Published by Elsevier Ltd.)

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Corneal transparency is fundamental to the visual system, and is directly related to the ordered collagen fibril architecture that the cornea maintains. Proteoglycans, through their protein core and highly anionic glycosaminoglycan side chains, are thought to regulate the collagen organisation in the corneal stroma. To understand the inter-relationships between proteoglycans and collagen fibrils in the cornea, adult mouse corneas were treated with cuprolinic blue and three-dimensional reconstructions of the anterior, mid and posterior corneal stroma were obtained. The reconstructions show regular diameters of collagen fibrils throughout the cornea and uniform interfibrillar spacing within each region. Both longitudinal and transverse reconstructions were obtained to establish a clear picture of proteoglycan organisation, yet no distinct regular pattern or symmetry of proteoglycan orientation was observed. Large, electron-dense proteoglycans (possibly chondroitin sulphate/dermatan sulphate proteoglycans) interconnecting two or often three adjacent collagen fibrils are seen, whilst another sub-population of smaller proteoglycans (of the keratan sulphate variety) interconnect only neighbouring fibrils. The reconstructions suggest a complex interaction between proteoglycans and collagen, which allows for the dynamic control of collagen fibril architecture in the cornea., (Copyright 2010 Elsevier Inc. All rights reserved.)

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.