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 (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, , 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 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 (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 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% 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 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 (TEM); some contributing to allosteric interactions between neighbouring chan- 37 examples are presented in Figure 1. nels. Thin section TEM of cardiac junctions has reported a reasonably Microarchitecture of the dyad 171 Downloaded from https://academic.oup.com/cardiovascres/article/98/2/169/278625 by guest on 23 September 2021

Figure 2 CryoEM map of RYR2 tetramer as viewed from the myoplasm (A), and from the side (B). TM, transmembrane stalk; MY, myoplasmic rectangular prism, circled regions highlight the domains forming the clamp. (C) Two tetramers showing the expected interaction via the clamp domain. regular spacing between the tetramers, comparable with that of skeletal ventricle myocardium has shown a comparable result, with numerous muscle triads,38 and coupled gating between RYR2s has been observed smaller and irregularly shaped clusters of RYR2 averaging only 14 tet- in vitro.39 Because of this and the structural similarity to RYR1, RYR2 has ramers per cluster, with edge-to-edge intercluster distances often also been thought to form a checkerboard lattice. However, recent ,50 nm.49 Neither study could resolve the distribution of individual electron tomographic analysis of rat ventricular myocytes fixed in situ tetramers within a cluster, although the latter used the checkerboard has shown RYR2 distributed in a combination of checkerboard and lattice as an a priori assumption with which to fit the data. side-by-side configurations with some isolated tetramers.40,41 This These new values represent a considerable drop from the previous surprising arrangement seemed to be a combination of two RYR1 orien- estimate of 75–100 tetramers/cluster.38,50 It has been difficult to + + tations observed in lipid bilayers, a checkerboard array at 0 mM Mg2 explain why a Ca2 spark arises from roughly 7–20 tetramers, but and a side-by-side orientation, where the tetramers were not in the new observations indicate why this might be so, as this seems + + direct contact, at 4 mM Mg2 .42 Since the in vivo Mg2 concentration to be the approximate size of a cluster. However, this does not + is 1 mM, it could be that the in situ distribution is an intermediate explain why Ca2 released from one cluster does not trigger neigh- arrangement between these two extremes. boring clusters within the same dyadic cleft given the very small dis- Part of domain 10 of the mouse RYR2 (residues 2699–2904) has tances between them. This question is currently being investigated.51 recently been crystallized and its structure solved at 1.65–1.95 A˚ Centre-to-centre distances between 300 and 750 nm were found resolution.43 This region hosts the phosphorylation sites S2808 and between RYR2 clusters using TEM and diffraction-limited fluores- S2814 (human numbering), whose physiological and pathophysiologic- cence microscopy,38,50 whereas in the tomograms a large fraction al effects are the subject of intense controversy.44 –46 This study iden- are much closer than that. This raises the question of how close tified five phosphorylation sites for PKA (S2797, S2808, T2810, S2811, two clusters can be and remain functionally independent. and S2815) and four for CaMKII (S2808, S2811, S2814, and T2876), These new data support a model of the dyad in which the RYR2 are some of which overlap. A large-scale analysis of phosphorylated pro- distributed in a number of small clusters including single receptors, teins in the mouse has identified all of these residues (except T2876), with the tetramers adopting multiple arrangements; this is dia- as well as T2781, Y2821, and S2822, as targets for in vivo phosphoryl- grammed and contrasted with our earlier view of RYR2 distribution ation.47 The analyses demonstrate that multiple sites can be phos- in Figure 3. The functional implications of this structural organization, phorylated simultaneously, raising the possibility that functional and how the Cav1.2 are distributed relative to the RYR2, have yet to effects could be cumulative and tuned by the number of sites involved. be determined. Tomographic data of mouse ventricle has provided surprising 48 insights into the architecture of the dyad. RYR2 were thought to 4. Ca 1.2 fill the dyadic cleft, but occupy only 78% of it. The average v volume of the dyadic cleft is an order of magnitude lower than previ- Cav1.2 is a heterotetramer composed of a pore-forming a1C subunit, ously thought (2.9 × 105 vs. 1.5 × 106 nm3 38), ranging from a one of the four b subunit isoforms which determine where in the 3 3 6 3 minimum of 9.3 × 10 nm to a maximum of 3.9 × 10 nm . Assum- plasmalemma the a subunit is targeted, an extracellular a2d subunit ing a 12 nm wide cleft and side-by-side packing,48 the maximum and a g subunit.52 Immunofluorescence has indicated that 75% of 53 number of RYR2 that each cleft could contain ranges from one to a Cav1.2 clusters in the rat ventricle are located in couplons. They maximum of 385, with a mean occupancy of 43, while checkerboard are smaller than the RYR2 clusters, and within the limits of diffraction- packing gives a maximum of 226 and a mean of 25. The median was limited microscopy, appear to be positioned over their centres.53 only 25 (15 checkerboard), indicating a distribution skewed by Functional analyses have suggested a tight physical association 2+ some large clefts. Super-resolution immunofluorescence of rat between Cav1.2 and RYR2. In these studies, Ca channel agonists 172 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 3 Contrast between the previously held (A) and newly developed (B) views of RYR2 distributions within the dyadic cleft. jSR is shown in outline (light patches) with their complement of RYR2. t-lumen of T-tubules.

+ + increased, and antagonists decreased, the Ca2 spark frequency in voltage-gated Na channels and NCX have functional access to the + permeabilized cells when there was no transmembrane Ca2 gradient dyadic cleft, but cannot discern whether they are nearby or physically 54,55 or flow through the channel. This implies that Cav1.2 affects RYR2 within it. Immunofluorescence studies in ventricular cells have pro- gating through a direct physical interaction or through intermediary vided conflicting results, with co-localization values for NCX–RYR2 63 –65 66 proteins. In support of this view, the C-terminus of the a1C subunit reported as ranging from 3–10 to 42%. Triple binds directly to RYR2 with high affinity, 6 nM, and injecting it into co-localization has been performed in rat atrial cells, and has demon- 2+ 56,57 atrial cells increases Ca spark frequency. These data suggest strated a statistically significant number of Cav1.2-RYR2-NCX triples 67 that Cav1.2’s location within the couplon is closely tied to that of comprising 13% of the RYR2 and NCX clusters. These experi-

RYR2. Additional details of Cav1.2 clustering and the factors that ments also demonstrated a much tighter association between 58 67 control and affect it can be found in a recent review. Cav1.2 and RYR2 than between NCX and RYR2–Cav1.2, implying that NCX is at the periphery of the RYR2–Cav1.2 clusters. These 1 21 co-localization values are also consistent with NCX currents in pig 5. The Na /Ca exchanger myocytes that have demonstrated that 10–15% of NCX current + 2+ 68 The Na /Ca exchanger (NCX) is a secondary active transporter, (INCX) is in or near the dyadic cleft. Unpublished results from our + + translocating three Na ions in exchange for one Ca2 ion across laboratory indicate that the molecular architecture of the ventricular + the plasmalemma, and is the main route for Ca2 extrusion from all dyads are similarly diverse. myocardial cells.59 The exchanger is reversible, with the direction of NCX is anchored into the membrane, in part, by ankyrin-B, a multi- + + + + transport being determined by the local Na and Ca2 gradients as valent adaptor protein that also binds and localizes the Na /K well as the membrane potential. The reversibility has led to the pro- ATPase.64,69 Multiple experiments have demonstrated that the activ- + posal that under some circumstances NCX can bring Ca2 into the ities of the ouabain-sensitive a2 isoform of NKA and NCX are cell and directly affect, or initiate, ECC. The strongest support for tightly coupled.69 –71 These data demonstrate that the a2 isoform + + this hypothesis has come from the cardiac-specific NCX knock-out of NKA can affect Na and Ca2 handling within the dyad and alter (KO) mouse.60 A comparison of ECC in the KO and wild-type ECC, but the exact position relative to the dyad is unknown.

(WT) has demonstrated that current through the voltage-gated A more detailed discussion of Cav1.2 and NCX, their positions + + Na channel (INa) increases the local Na concentration reversing relative to the dyad and the functional implications of the molecular NCX and augmenting ECC, an effect completely absent in the KO architecture, has recently been published.58 mice. In these experiments, NCX could not initiate ECC, but the The myoplasmic domain of RYR2 is reported to scaffold at least 17 + Ca2 it brought into the cell ‘primed’ the RYR2 and acted synergistic- molecules that affect its activity. This includes kinases and their 2+ 61,62 ally with Ca entering through Cav1.2. These data indicate that anchoring proteins, calmodulin, FKBP12, and 12.6, and soluble Microarchitecture of the dyad 173

In skeletal muscle, there is evidence that JPH1 is linked to both 77 Cav1.1 and RYR1, but the exact relationship in cardiomyocytes is unclear; super-resolution images of JPH2 and RYR2 show differing dis- tributions that vary from dyad to dyad, ranging from each RYR2 having a JPH2 adjacent to it, to having the JPH2 at the periphery of the dyad surrounding all of the RYR2 within it.78 In mice in which knock-down of JPH2 was achieved using tamoxifen-induced siRNA, there was a 40% reduction in the number of dyads.79 The width of the remaining dyadic clefts was, on average, no different than control, but displayed more variance, even within a single dyad (1.96 nm2 in siRNA-treated mice vs. 1.21 nm2 in the controls). The functional consequences were

a significant reduction in Cav1.2 co-localization with RYR2 and in the Downloaded from https://academic.oup.com/cardiovascres/article/98/2/169/278625 by guest on 23 September 2021 + + amplitude of the Ca2 transient, but no change in the Ca2 current. These results suggest that JPH2 is not only responsible for maintaining

the dyadic structure but was keeping the Cav1.2 positioned opposite the RyR2, a role similar to that in skeletal muscle. This would be con- sistent with recently reviewed functional and structural evidence

pointing to a much closer physical association between Cav1.2 and RYR2 than previously thought.

7. The RYR macromolecular complex within the SR Figure 4 Diagram depicting a single junctophilin-2 molecule con- necting the plasmalemma and junctional SR membranes within the RYR2 forms a macromolecular complex with a number of other mole- dyad. Adapted from Garbino114 with permission from Macmillan cules in the SR; the SR transmembrane proteins triadin and junctin, Publishers Ltd. whose N-terminals extend a short way into the dyadic cleft and two SR-bound calcium storage molecules; calsequestrin (CASQ), a high 80 proteins like sorcin that can modify the channel’s flux. This review will affinity but low capacity calcium binder and histidine-rich calcium- be limited to those that are important in determining the structural binding protein (HRC) which has a high capacity, but low affinity, for 81 properties of the dyad, a complete description and analysis of the calcium. Both of these storage molecules interact with junctin and others can be found in recent reviews.33,34,46 triadin which, in turn, affect the ryanodine receptor and thus the behaviour of the dyad. 82 83 6. Junctophilin Junctin and triadin have similar structures; the C-terminal domain of both molecules binds to RYR2 and CASQ forming a macro- An essential component of the dyadic cleft is the molecule junctophi- molecular complex. Triadin has disulphide bonds allowing it to form lin 2 (JPH2), one of a family of four isoforms which are present in the oligomers, and it is in this state that it anchors CASQ to the membrane junctional membrane complexes of other excitable cells such as skel- of the jSR. It was initially thought that there were three cardiac triadins etal muscle (JPH1) or brain (JPH3 and 4).72,73 JPH2 is a 74 kDa mol- (CT1-3), with molecular weights of 35, 40, and 95 kDa, respectively.83 ecule which spans the cleft from the plasmalemma to the SR, and plays CT2 was later identified as glycosylated CT1,84 and CT3 is ,5% of the the central role in the development and maintenance of that space. total. A later analysis of mouse cardiac triadin reported three splice The end touching the plasma membrane has 8 MORN (Membrane variants with molecular weights of 35, 35.5, and 40 kDa, and their func- On Receptor Nexus) sequences while the other end, the transmem- tional significance remains unknown.85 brane domain, is embedded in the SR. The MORN sequence is highly Although triadin-null mice appear to be normal and grow to matur- conserved across species74 and seems to be responsible for binding ity, such mice have fewer dyads with a smaller extent and less RYR2 both to lipids and proteins in the t-tubular membrane. The section than do wild-type.86 These animals have reduced co-localization spanning the dyad cleft consists of an alpha-helix as well as a divergent between Cav1.2 and RYR2, a reduced CASQ expression and an region that is associated with the characteristics of each isoform almost complete absence of junctin. In addition, they are susceptible (Figure 4). There are a number putative phosphorylation sites on to catecholaminergic polymorphic ventricular tachycardia (CPVT) + JPH2, but none have been associated with a particular function.74 due to an increased SR Ca2 leak. Recently, it has been shown that JPH2 KO mice are embryonic lethal and fail at about the time the individuals who suffer mutations in triadin also suffer from CPVT heart beat is established (E10.5); EM imaging shows that the distance resulting in sudden cardiac death.87 Junctin-null mice appear to have + between the plasmalemma and the membrane of the SR was few structural alterations and no changes in any other Ca2 handling increased compared with control embryos,73 indicating that keeping proteins, but are more susceptible to CPVT and die very young after the two membranes a fixed distance apart was a major function of as little as 5 weeks of life.88 The difference with the triadin-null mice is this molecule. The width of the dyadic cleft is critical not only for remarkable given their similarities. effective CICR, but also for calcium-dependent inactivation (CDI) of Junctate, like junctin, is a splice variant of aspartyl beta-hydroxylase, 2+ Cav1.2, whereby the Ca released from RYR2 inactivates Cav1.2 to but the differing charges on their C-terminals, positive for junctin and speed the rate of repolarization.75,76 negative for junctate, mean that they serve different functions: junctin 174 D.R.L. Scriven et al.

2+ interacts with RyR and CASQ/HRC while junctate interacts with Ca coupling between Cav1.2 and RYR2 in hypertrophied hearts with and InsP3R89 and SERCA2a.90 Because of its binding partners, it is un- no appreciable change in the cellular architecture, the density and + likely that junctate is situated in the dyad portion of the SR membrane. function of either channel, or the SR Ca2 load, any of which might Calsequestrin is a monomeric glycoprotein with three highly acidic have produced the same effect. Recent data from studies of + pockets that bind Ca2 . The cardiac form (CASQ2) appears to be pressure-overloaded Sprague–Dawley rats support this hypothesis, mostly in the form of monomers or dimers at physiological levels and suggest that a reduction in the expression of JPH2 precedes the 91,92 of SR calcium, which contrasts with skeletal muscle where it loss of Cav1.2–RYR2 intercommunication on which both CICR and forms more complex linear polymers when binding millimolar CDI depend.102,103 A loss of JPH2 would be expected to ‘unglue’ + + Ca2 .80 It is well established that Ca2 release through RYR2 is a non- the jSR from the T-tubules and widen the dyadic cleft and this has + linear function of the luminal Ca2 concentration, which implies that been observed in a pressure-overloaded model of failure.104 The 2+ the channel is controlled either directly by Ca or indirectly via initial uncoupling of RYR2 from Cav1.2 increases the distance

CASQ. There is evidence for both mechanisms, but which of the between them, which should reduce the effectiveness of both CICR Downloaded from https://academic.oup.com/cardiovascres/article/98/2/169/278625 by guest on 23 September 2021 two, or a combination of both, actually controls the RYR2 open prob- and CDI, which may explain Go´mez’s observations. Additional evi- ability is still a matter of debate.93 CASQ mutations in humans are well dence for the involvement of JPH2 comes from people suffering known to have deleterious effects with CPVT and sudden cardiac hypertrophic cardiac myopathy who have reduced concentrations death being common outcomes.94 CASQ null mice have a larger SR of JPH2,105 and a probable mechanism for this reduction is the than normal; they are also subject to CPVT probably due to an up-regulation of microRNA, which reduces the expression of JPH2.106 increased SR leak as well as a reduction in control of the RYR2 At later stages of disease, the TATS undergoes extensive remodel- 95 Po. The width of the jSR is wider in the CASQ null mice compared ling eventually becoming fractured, convoluted, and disorganized, with the wild type. This characteristic is shared with the triadin nulls, leaving isolated and ‘orphaned’ RYR2. The latter become subject to + but the down-regulation of CASQ expression suggests that the excitation via Ca2 diffusion, similar to its neonatal state, leading to + changes in the jSR are a secondary effect. unsynchronized Ca2 release, delayed after depolarizations, electrical The other calcium-binding protein in the SR, the HRC, is, like malfunction, and death.107 – 110 CASQ, highly charged and is thought to exert its calcium-binding Other studies have shown that heart failure is associated with a properties through electrostatic interaction with pairs or triplets of reduction in the size of the dyad104 as well as a down-regulation of 111 amino acids. HRC binds with triadin through which it can modulate a slew of proteins, including CASQ, junctin, NCX, and Cav1.2; the behaviour of RYR2. HRC can also partner with SERCA, the the reduction in the latter two are probably the result of the + ratio of the two proteins being determined by the luminal Ca2 con- reduced t-tubular volume. It has also been shown that + centration. In rat ventricular tissue, the amount of Ca2 binding to propagation in the TATS of rats suffering heart failure regularly fails HRC constitutes a significant portion of the total.92 HRC has a com- even if the tubules remains attached to the sarcolemma.112 pensatory up-regulation when CASQ is ablated,92 probably to provide + additional Ca2 buffering. 10. Summary 8. Development of the dyad In little over a decade, our understanding of dyad architecture has In mammals, the T-tubules are absent at birth; in the rat, ventricular car- been conceptually transformed from one in which the cleft was diomyocytes have no TATS at Day 10 but it is fully present by Day 20.96 filled with hundreds of RYR2 tetramers arranged in a continuous Electron microscope observations show that very little dihydropyri- checkerboard array forming a single couplon to one where dyads dine receptor appears opposite the RYR early in development.97 are both structurally and functionally diverse. Observations indicate This fits well with the observations of developing rabbit myocardium that they contain multiple RYR2 clusters of varying sizes and tetramer where the peripheral couplings have NCX co-localized with RYR2 organization and are associated with different channels and transpor- and reverse-mode calcium influx is the method whereby ECC is ters that alter their function. Some RYR2 clusters do not have Cav1.2 53,65,113 initiated.63 As the animal matures, NCX expression declines98 while opposite them and are therefore not in couplons, suggesting the number and density of NCX clusters co-localized with RYR2 that some dyads may not contain a couplon or that not all clusters decreases.63 In parallel, there is an increased co-localization of RYR2 within a dyad form couplons. Conversely, some dyads might house 99 many couplons, producing additional functional diversity. With the ap- with Cav1.2, producing the adult form of CICR where ICa initiates + Ca2 release from RYR2. The first molecule that appears in the dyad plication of new imaging techniques and the discovery of new mole- is probably JPH2 which anchors the SR to the plasmalemma, then cules that support and modulate dyad structure and function, CASQ enters the SR lumen with triadin and junctin following shortly long-held hypotheses are being challenged. The functional implications thereafter.97 A study of developing T-tubules in the rat ventricular car- of these discoveries are just beginning to be explored, but are likely to diomyocyte suggests that the TATS is generated in two ways; the ma- produce a new theoretical foundation from which to interpret the jority penetrating into the cell along Z the lines while other tubules functioning of the myocyte in both health and disease. expand from small pockets of membrane containing Ca 1.2 that form v Conflict of interest: none declared. within the cell interior and eventually join existing RYR2 clusters.100

9. Structural alterations in disease Funding This work was supported by the Canadian Institutes of Health Research 101 Go´mez et al. were the first to hypothesize that contractile failure (MOP-115158); and the Natural Sciences and Engineering Research followed a structural change within the dyad. They found reduced Council of Canada. Microarchitecture of the dyad 175

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