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Microarchitecture of the Dyad Cardiovascular Research (2013) 98, 169–176 SPOTLIGHT REVIEW doi:10.1093/cvr/cvt025 Microarchitecture of the dyad David R.L. Scriven, Parisa Asghari, and Edwin D.W. Moore* Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 Received 12 December 2012; revised 2 February 2013; accepted 4 February 2013; online publish-ahead-of-print 11 February 2013 Downloaded from https://academic.oup.com/cardiovascres/article/98/2/169/278625 by guest on 23 September 2021 Abstract This review highlights recent and ongoing discoveries that are transforming the previously held view of dyad structure and function. New data show that dyads vary greatly in both structure and in their associated molecules. Dyads can contain varying numbers of type 2 ryanodine receptor (RYR2) clusters that range in size from one to hundreds of tetramers and they can adopt numerous orientations other than the expected checkerboard. The association of Cav1.2 with RYR2, which defines the couplon, is not absolute, leading to a number of scenarios such as dyads without couplons and those in which only a fraction of the clusters are in couplons. Different dyads also vary in the transporters and exchangers with which they are associated producing functional differences that amplify their structural diversity. The essential role of proteins, such as junctophilin-2, calsequestrin, triadin, and junctin that main- tain both the functional and structural integrity of the dyad have recently been elucidated giving a new mechanistic understanding of heart diseases, such as arrhythmias, hypertension, failure, and sudden cardiac death. ----------------------------------------------------------------------------------------------------------------------------------------------------------- Keywords Dyad † Structure † Ryanodine receptors † Morphology † Molecular architecture ----------------------------------------------------------------------------------------------------------------------------------------------------------- This article is part of the Spotlight Issue on: T-tubules and ryanodine receptor microdomain signalling in cardiac hypertrophy and failure. + + of Ca2 triggers a much larger Ca2 release into the myoplasm 1. Introduction + through the RYR2 in the jSR, a mechanism of ECC called Ca2 - + Our understanding of how both familiar and newly identified mole- induced Ca2 release (CICR).6,7 Even though CICR is a process of cules contribute to the structure and function of the dyad has been positive-feedback, an open RYR2 does not trigger all of the others transformed in recent years. The function of these molecules, and leading to an explosive all-or-none event. their role in health and disease, has been elucidated using genetically This problem, dubbed the calcium paradox, was first solved theor- modified animals and a number of new techniques. This review will etically by positioning RYR2 into groups of physically and functionally highlight recent developments in the field, while noting that earlier isolated clusters that were independently controlled.8 Experimental reviews have detailed the fundamental observations and hypotheses support for this hypothesis followed rapidly. The first were observa- 1– 5 + which laid the foundation for our current progress. tions describing Ca2 ‘sparks’, which are the result of stochastic open- + We will refer to the T-tubules as the transverse-axial tubule system ings of a small cluster of RYR2 that do not lead to all-or-nothing Ca2 (TATS) since it contains many axial as well as transverse elements, transients.9– 13 These were spatially isolated events encompassing a + while the term sarcolemma will be restricted to the cell surface; large volume that did not trigger Ca2 release from other dyads. Fol- plasmalemma will refer to both. This terminology reflects the lowing this, electron microscopy and immunofluorescence analyses need to distinguish between these areas of the membrane despite identified clusters of Cav1.2 in the plasmalemma-situated opposite forming a continuous structure. The term dyad refers to the structural clusters of RYR2 in the jSR; the two being separated by + element formed by the close apposition of the sarolemma and the 15 nm.14,15 The Ca2 sparks and the structural analyses provided + junctional sarcoplasmic reticulum (jSR), while a couplon is the func- the basis for local control theory, which states that Ca2 release tional element within the dyad formed by juxtaposition of type 2 rya- from one couplon does not affect the others because of the physical + nodine receptors (RYR2) in the jSR with cardiac L-type voltage-gated separation between them and the high local Ca2 required to activate 2+ Ca channels (Cav1.2) in the adjacent plasmalemma. RYR2. ECC and local control theory are discussed in detail by Bers.16 + + The fundamental event in ECC is a Ca2 spark, and a Ca2 transient is 2. Structure of the dyad and local the macroscopic sum of thousands of sparks. This enables the force of contraction to be a function of the number of sparks that are initiated, control of ECC equivalent to the number of couplons that are recruited. At the time Excitation–contraction coupling (ECC) is initiated by an influx of these theories were formulated a dyad was thought to contain a single 2+ Ca through the Cav1.2 on the plasmalemma. This small influx large couplon, but new discoveries require that some of these ideas * Corresponding author. Tel: +1 604 822 2316; fax: +1 604 822 3423, Email: [email protected] Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2013. For permissions please email: [email protected]. 170 D.R.L. Scriven et al. Downloaded from https://academic.oup.com/cardiovascres/article/98/2/169/278625 by guest on 23 September 2021 Figure 1 Transmission electron micrographs of rat ventricular myocytes fixed in situ displaying junctions with various morphologies. (A) Junction encircles T-tubule; (B) sarcolemmal junction; (C) dyad; (D) triad; (E) junction on longitudinal tubule; (F) surface junction at intercalated disc. RYR2, double arrow; CASQ, arrow; ec, endothelial cell; is, interstitial space; jSR, junctional sarcoplasmic reticulum; t, T-tubule; m, mitochondria; z, z-line. Insets are two-fold magnifications of the boxed regions. Scale bars ¼ 50 nm. Reprinted from Asghari et al.20, with permission from Elsevier. be re-examined. However, the concept that CICR is a result of, and is 3. Ryanodine receptors dependent on, the molecular composition, architecture, and distribu- tion of the dyads remains unchallenged. The RYR2 gene encodes a large 565 kDa protein that forms homote- 2+ Dyads are found throughout mammalian myocytes with a well- tramers creating RYR2, the SR Ca release channels whose molecular developed TATS, which includes the ventricles, and the atria of weight is 2.2 MDa. Mammals express three isoforms (RYR1-3), with 22 – 25 larger species.17,18 The presence of a TATS seems to be determined the working myocardium expressing only RYR2. The isoforms are by the size of the myocyte; even in small mammals the larger atrial highly homologous, with rabbit RYR2 sharing 70% amino acid identity 22,23 myocytes are more likely than not to have a rudimentary TATS with RYR1 and RYR3. Cryo-electron microscopy of RYR1 demon- when the cells are greater than about 10 mm wide.19 Myocytes strates that 80% of the protein extends into the myoplasm in the without a TATS have corbular SR (extra-junctional SR), which are shape of a rectangular prism (29 × 29 × 13 nm) and has multiple 26 – 28 sacs of SR decorated with RYR2 projecting into the myoplasm. solvent-accessible cavities. More recent work has revealed large + These are positioned along the Z-line and can release Ca2 in allosteric movements associated with RYR1 gating, particularly in the + response to Ca2 diffusion from sarcolemmal dyads, functioning as clamp region (domains 5–10) which moves 8 A˚ towards the surface 29 a secondary amplification system. The jSR always appears as a of the SR. The three isoforms have near identical three-dimensional narrow sac that is relatively flattened when adjacent to the sarco- structures, as expected from the large sequence similarity, so compar- lemma, but it can adopt a number of configurations around the able gating movements are expected from RYR2. Additional structural 30 –34 TATS, forming dyads, triads, and more complex geometries, including details are available from recent reviews. junctions that span an entire sarcomere.20 The lumen of the jSR con- In skeletal muscle triads, RYR1 are distributed in a checkerboard + tains electron dense material, which is largely composed of the Ca2 - array whereby each tetramer contacts its neighbours through 35 binding protein calsequestrin (CASQ).21 The surface of the jSR facing domains 6 and 10 in the protein’s clamp region, Figure 2. In vitro experi- the plasmalemma is populated by electron dense projections that ments using purified RYR1 inserted into lipid bilayers have shown that extend most of the width of the dyadic cleft. These are the RYR2, the same lattice forms spontaneously, demonstrating this to be an intrin- 36 also known as the foot processes. Their position and characteristic sic property of the molecule. Since the clamps undergo major con- shape make them one of the few proteins that can be positively iden- formational changes as the channel opens and closes, they are likely tified in standard transmission electron microscopy
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