Physiology and patho-physiology of the cardiac transverse tubular system

Short title: Transverse (t)-tubules in the heart

C.E.R. Smith, A.W. Trafford, J.L. Caldwell and K.M. Dibb*

Unit of Cardiac Physiology, Division of Cardiovascular Sciences, Manchester Academic Health Sciences Centre, Central Manchester Foundation Trust, University of Manchester, 3.14 Core Technology Facility, 46 Grafton Street, Manchester. M13 9NT. United Kingdom.

Correspondence to Katharine M. Dibb, PhD.

Tel: (+44) (0)161 275 1195

E-mail: Katharine.Dibb@manchester.ac.uk

Unit of Cardiac Physiology, University of Manchester, 3.14 Core Technology Facility, 46 Grafton St, Manchester. M13 9NT. UK.

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Abstract

Cardiac transverse (t)-tubules play a vital role in ensuring synchronous contraction. They contain L-type Ca2+ channels which closely couple with intracellular Ca2+ release channels throughout the cell to mediate a rapid and uniform Ca2+ release. The complexity of the t-tubule network varies between species and across cardiac chambers and is also highly labile with density increasing during development and decreasing in disease. Recent work using super-resolution and 3D electron microscopy has shown that t-tubules themselves are highly diverse structures with the proteins located on and around them differentially modulated compared to other sites within the cell. This review will summarise our current understanding of the t-tubule network in health and disease with focus on t-tubule heterogeneity, the importance of t-tubules in excitation-contraction coupling and the proteins responsible for t-tubule regulation.

1. Introduction

In cardiac cells a rise in intracellular Ca2+, referred to as the systolic Ca2+ transient, is responsible for the initiation of contraction. This rise in Ca2+ occurs when Ca2+ entry via L-type Ca2+ channels (LTCCs) triggers Ca2+ release through ryanodine receptors (RyRs) on the intracellular Ca2+ store, the sarcoplasmic reticulum (SR). This process of Ca2+ induced Ca2+ release (CICR), or triggered Ca2+ release, relies on the close association between LTCCs on the cell membrane and the RyRs of the SR, which together form the cardiac dyad, and ensure synchronous contraction. In the ventricle, during the systolic Ca2+ transient, Ca2+ rises rapidly and synchronously throughout the cell. This is achieved due to the presence of a highly regular network of transverse (t)-tubules. T-tubules are deep invaginations of the surface membrane, associated with the z-line of the sarcomere, which express a variety of ion channels and receptors including LTCCs (Figure 1). Thus LTCCs form dyads with RyRs along the z-line allowing Ca2+ release to be triggered throughout the t-tubule network as well as at the surface membrane. Figure 1 depicts a single t-tubule and associated Ca2+ handling machinery with membrane staining highlighting the t-tubule network and surface membrane in ventricular and atrial cells in Figure 2. Given this structural information it is easy to envisage how cardiac cells with a regular t-tubule network can achieve a rapid and synchronous rise in systolic Ca2+.

2. Distribution of t-tubules within the myocardium

Remarkably, while the size of the ventricle changes dramatically between small and large mammalian species, ventricular myocytes are uniform in size [1] (Table 1) and possess well-developed t-tubule networks (Figure 2A; Table 1). The structure of the ventricular t-tubule network has been examined in detail and consists of predominantly transverse elements which extend into the cell interior along the z-line together with some longitudinal extensions [2]. Compared to small mammalian species large mammals, including the human, possess a less complex ventricular t-tubule network consisting of wider t-tubules with fewer t-tubule branches and reduced network density [3-5] (Figure 2A).

Whereas ventricular t-tubule networks are relatively similar across species, there are striking differences in t-tubules in the atria between small and large mammals as summarised in Table 1. These differences are associated with increased atrial myocyte size in larger species (Figure 2B). Most, but not all [6,7], studies in the atria of small mammals document either a lack of atrial t-tubules e.g. [8,9] (Figure 2Bi; Table 1) or a sparse network of largely

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longitudinally orientated (axial) tubules often referred to as transverse axial tubules (TATs) [10,11] (Figure 2Bii). The density and organisation of atrial t-tubules depends upon the localization within the atria [6,7]. We [12,13] and others [7,14,15] have shown that atrial t-tubules in large mammals are fundamentally different to TATs. Here, large mammalian atrial t-tubules form a mainly transverse network which is similar in structure but less dense than in the ventricle (Figure 2Biii; Table 1).

3. Role of the t-tubule in Excitation-Contraction CouplingIn ventricular myocytes the density of LTCC current (ICa-L) is approximately 9 times higher on the t-tubule than on the surface membrane [16,17](Table 1). Immunocytochemistry shows LTCCs and RyRs are distributed almost completely along the z-line resulting in dyad formation at t-tubules with few peripheral couplings [18]. In healthy ventricular myocytes the SR forms a continuous network throughout the cell, wrapping around t-tubules and forming many discrete dyads which are variable in size and occur on both transverse and longitudinal t-tubule extensions [2,19]. Therefore the majority of triggered Ca2+ release occurs at the t-tubule in the ventricle. In addition to being key for CICR, t-tubules also play an important role in Ca2+ extrusion with the sodium-calcium exchanger (NCX) and plasma membrane Ca2+ ATPase (PMCA) predominantly active on t-tubules compared to the surface sarcolemma [20,21].

More recently the use of super-resolution microscopy and 3D electron microscopy techniques has shown the precise architecture of the t-tubule network highlighting that the dyad is not as uniform as we perhaps first thought. T-tubules in mouse have been shown to contain dense inner membrane folds which act as a diffusion barrier and effectively divide the t-tubule lumen into spatial subdomains [22]. Similar structures have been observed following myocardial infarction (MI) in the pig [23]. Non-uniform dilations have also been observed along t-tubules [2,4]. However how such changes in t-tubule structure alter the efficiency of triggered Ca2+ release remains to be determined [22]. Similarly to t-tubules, RyRs also appear to be heterogeneous in ventricular myocytes. As RyRs exist in clusters, factors such as cluster distribution, size and modulation all influence Ca2+ release and appear to vary throughout the cell. For example in the large mammalian ventricle, where t-tubules do not occur on every z-line, some RyR clusters are not coupled to membrane (as discussed for the atria below). Modulation of Ca2+ release e.g. by Ca2+/calmodulin-dependent kinase II differs between coupled and non-coupled RyRs [24]. RyR cluster size is also highly variable and RyR clusters associated with t-tubules are almost four times larger than those at the ventricular cell surface [25,26].

3.1 Role of atrial t-tubulesFar less is known regarding the precise structure of both t-tubules and dyads in the atria. In addition, the role of the atrial t-tubules in Ca2+ release will likely differ between cells and species given that gross t-tubule density and structure varies widely. Generally, compared to the ventricle, the reduced density of t-tubules in the atria increases the reliance of atrial cells on propagation of the Ca2+ signal to regions lacking t-tubules. In atrial cells which are devoid of t-tubules the initial rise in Ca2+ occurs only at the cell periphery and propagates to the cell centre as a wave of CICR [9,27]. We and others have shown that both TATs in rodents and t-tubules in large mammals are able to trigger Ca2+ release in the interior of atria myocytes [7,10,12], suggesting the presence of dyads at these triggered sites. Super-resolution scanning patch clamp shows that, in contrast to the ventricle, LTCCs distribute equally between the t-tubule and surface membrane in the atria [6]**. Despite this, in the atria not all Ca2+ release sites are created equally. ‘Eager’ Ca2+ release sites open rapidly on the cell surface and have a

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fixed location and activation order [27]. Whereas in cells with TATs specialized structures exist on the longitudinal extensions of the t-tubule system. Here highly phosphorylated RyR clusters result in triggered Ca2+ release occurring twice as fast at these ‘super hub’ sites compared to release sites at the surface membrane [28]**. Whether specialized Ca2+ release sites exist on large mammalian atrial t-tubules remains to be determined. Interestingly, in human, LTCCs on the t-tubule have higher open probabilities than channels on the surface membrane [6]** suggesting large mammalian atrial t-tubules may also represent specialist sites for Ca2+ release.

4. The labile nature of t-tubulesDespite the extensive nature of the t-tubule network and their importance in facilitating triggered CICR, t-tubules are highly labile structures thus allowing cells to adapt to changing demands. T-tubule loss has been noted in the ventricle under diverse situations including culture [29] and in pathology [30-32]. Moreover, t-tubule density increases during postnatal development [33], exercise [34] or following SERCA gene therapy in heart failure (HF) [35]. Similarly to the ventricle, atrial t-tubules are lost in numerous disease states [12,14,36] and we have shown this loss is almost complete in HF [12]. The loss of atrial t-tubules in HF is associated with a decrease in ICa-L which is responsible for a reduction in the atrial systolic Ca2+ transient [37] highlighting the importance of the atrial t-tubule network. In light of this, the restoration of t-tubules in disease represents a promising therapeutic strategy.

4.1 Neonatal t-tubule developmentThe labile nature of t-tubules is also highlighted during their formation in the neonate myocardium. In the ventricles of small mammals, t-tubules are absent at birth and develop postnatally (Figure 3A; Table 1) with subsequent changes in excitation-contraction coupling [33,38,39]. As a consequence of a lack of t-tubules in the neonate ventricle, LTCCs are confined to the surface sarcolemma resulting in reduced ICa-L density and impaired LTCC coupling with RyRs [38-41]. Because of this, the systolic Ca2+ transient is slower to rise and reduced in amplitude in early life compared to adult [33,38,40,42]. The reduced coupling between LTCCs and the SR in neonates is reported to be compensated for by reverse mode operation of NCX [43,44], with Ca2+ influx via NCX alone able to trigger contraction in the newborn rabbit ventricle [45]. As t-tubules develop the need for NCX-mediated Ca2+ influx is diminished as increased LTCCs found on t-tubules trigger synchronous SR release resulting in a rapid uniform rise of systolic Ca2+ [39,40].In contrast to small mammals, t-tubules have been observed prior to birth in the foetal sheep and human ventricle (Table 1), suggesting possible species related differences in the onset of t-tubule formation [46,47]. The reasons for these differences are not fully understood but are likely to relate to species variation in developmental maturity at birth. Whilst t-tubules are present at birth in large mammals they continue to develop postnatally [46]*, indicating that whilst the time frame of formation is shifted the process appears to be unaltered between species.

4.2 T-tubule remodelling in diseaseIn HF many studies have reported disordered ventricular t-tubules with a general increase in longitudinal elements [29,48,49], sheet-like remodelling [50]** and/or a patchy or moderate loss of t-tubules [30-32,51,52] resulting in a delayed and dys-synchronous rise in systolic Ca2+

[31,50,52] (Figure 3B; Table 1). Thus t-tubule loss and disorder in disease has been associated with contractile dysfunction in the ventricle [31,51,53]. Even though many t-tubules remain in the ventricle during HF, dys-synchronous Ca2+ release is exacerbated by failed action potential propagation into pathologically remodelled t-tubules [54]. Moreover,

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in HF some t-tubules move away from z-line structures thus leaving behind RyRs separated from LTCCs on the t-tubule membrane. These ‘orphaned’ RyRs contribute to dys-synchronous Ca2+ release and have been implicated in the genesis of Ca2+ dependent arrhythmias [48]. In disease the modulation of coupled and un-coupled RyRs is also altered [24] however whether this plays a role in arrhythmogenesis is yet to be determined. Interestingly, while mechanical unloading can improve function in some HF patients, in those with a high degree of t-tubule remodelling, resulting in increased RyR-membrane distances, mechanical unloading was not effective [50]**. Thus a largely intact t-tubule network may be necessary for cardiac recovery under these conditions [50]**. In terms of ICa-L, in the ventricle HF results in a redistribution of LTCCs from the t-tubule to the crest of the sarcolemma where open probability is increased [17]. We and others report an overall decrease in ICa-L in HF e.g. [55,56] however others report no change [31,57]. This discrepancy may be due to differential regulation of LTCCs by, for example, phosphorylation in HF.

Compared to the ventricle less is known regarding remodelling of atrial t-tubules in disease, however reduced atrial t-tubule density is observed in HF, atrial fibrillation (AF) and MI [6,12,14,58] (Table 1). It appears that like in the ventricle, atrial t-tubule remodelling correlates with the extent of disease whereby t-tubules switch to a more longitudinal orientation in hypertrophy [28]** but are almost entirely lost in HF [12]. Thus in HF atrial t-tubule loss is more severe than that in the ventricle [12,59] (Figure 3B). The mechanism for this extensive t-tubule loss is unknown but it is conceivable that the relatively thin walled atria are more susceptible to wall stress, arising as a consequence of ventricular dysfunction. As in the ventricle a number of studies report a decrease in atrial ICa-L in disease [14,37,60]. T-tubule loss is likely to play a role in reduced ICa-L [14] but in addition the reduced amplitude of ICa-L on remaining t-tubules is also likely to be important [6]**. The decrease in ICa-L observed in the sheep atria (mentioned above) is responsible for a decrease in Ca2+ transient amplitude [37] which, like in the ventricle, may also contribute to arrhythmias. For example decreased ICa-L is a hallmark feature of AF where it plays a role in action potential shortening and therefore shortening of the atrial effective refractory period important for the activation of re-entrant circuits involved in AF (reviewed in [61]).

5. Proteins responsible for t-tubule regulationAt present the proteins and pathways underlying t-tubule formation or loss in disease are not fully understood but are the subject of ongoing research. During t-tubule development initial invaginations at the cell surface are thought to be reliant on either translocation of proteins from the sarcolemma along the t-tubule or the fusion of vesicles to the membrane [62]. A number of proteins have been implicated in t-tubule development (Figure 1). Firstly, Amphiphysin II (AmpII) is known to be important for inducing membrane curvature and has been shown to induce tubule formation in non-muscle cells [63,64]. Junctophilin 2 (JPH2) which spans the dyad and is important for maintaining dyad structure and the close association between LTCCs and RyRs has also been implicated as being necessary for t-tubule maturation [65,66](reviewed in [67]). Both AmpII and JPH2 expression increase postnatally and knockdown of either protein results in reduced t-tubule density and increased disorder leading to smaller, less synchronous Ca2+ transients [22,59,65,66]. While JPH2 has also been shown to colocalise with developing t-tubules [38,46]* a recent study shows it only plays a minor role in this process [68]**. This study uses a novel approach designed to investigate JPH2 inactivation whilst avoiding the secondary effects of cardiac dysfunction and reveals JPH2 is not required for overall t-tubule formation but rather to stabilize t-tubules during cardiac dysfunction [68]**. Additionally this work identifies RyR2 as an important regulator of t-tubule maturation although the mechanism is yet to be determined [68]**.

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Given the discussion above it is perhaps not surprising that decreases in both AmpII and JPH2 have been associated with t-tubule loss in disease [51,59,69]. In addition telethonin (TCAP) is established as being important for normal t-tubules [70]. AmpII is thought to play a role in t-tubule loss in disease [59] and as stated above, JPH2 is required to stabilize t-tubules in the failing heart [68]**. AmpII also regulates the organisation of both LTCCs and RyRs within the dyad and therefore reduction of AmpII in HF has been suggested to impair this microdomain facilitating contractile dysfunction and arrhythmias [64,71]. TCAP resides at the z-disk (Figure 1) where it is thought to be a load dependent regulator of t-tubule structure with both unloading and overloading resulting in t-tubule disruption (reviewed in [72]). Both AmpII and JPH2 are promising candidates for therapeutic modulation. For example levels of AmpII correlate with cardiac function and can also predict future arrhythmias in patients without severe HF [73]. JPH2 gene therapy prevents t-tubule loss and maintains cardiac function in mice with early stage HF [74]*.

6. ConclusionsThe cardiac t-tubule is integral to both normal function and dysfunction in disease. The ventricular t-tubule has been extensively studied and only more recently has the atrial t-tubule become the focus of similar research. Thus our knowledge of the atrial t-tubule still lags behind that of the ventricle and this is exacerbated by the diverse nature of these structures in the atria. Recent work, using improved resolution imaging modalities, has revealed the intricate structure of t-tubules and dyads and how changes to structure alters Ca2+ release. This is highlighted in disease states where t-tubule disorder impairs Ca2+ release in both the atria and ventricle.

7. Future DirectionWhile some inroads have already been made into understanding the causes of t-tubule disorder and loss in disease this will also be an important avenue for future research. Understanding the pathways that regulate t-tubule formation and loss is an exciting area of research, the ultimate aim of which is to develop a method to restore t-tubules in disease with the hope that this will improve cardiac function in patients. Much work is needed before this can become a reality. Research is hampered by the fact that t-tubule remodelling is intricately linked with HF itself making it difficult to determine if remodelling is a cause or consequence of disease. Furthermore, in the atria, the diversity of the normal t-tubule network makes understanding remodelling more difficult. That said new models, discussed above, have been developed allowing exploration of the role of specific proteins in t-tubule formation without the complication of disease. The possibility of t-tubule restoration utilising proteins with multiple roles in t-tubule structure and Ca2+ handling may be key to producing a functional t-tubule network in patients where disease has not progressed to an irreversible stage. Therefore future research in this area is likely to be exciting and may open up new therapeutic strategies.

Acknowledgments

Work in the authors’ laboratories is funded by the British Heart Foundation.

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sites at discrete locations on TATs indicates spatial differences in RyR modulation around certain TAT structures.

[29] W.E. Louch, V. Bito, F.R. Heinzel, R. Macianskiene, J. Vanhaecke, W. Flameng, K. Mubagwa, K.R. Sipido, Reduced synchrony of Ca2+ release with loss of T-tubules-a comparison to Ca2+ release in human failing cardiomyocytes, Cardiovasc Res. 62 (2004) 63-73.[30] J. He, M.W. Conklin, J.D. Foell, M.R. Wolff, R.A. Haworth, R. Coronado, T.J. Kamp, Reduction in density of transverse tubules and L-type Ca(2+) channels in canine tachycardia-induced heart failure, Cardiovasc Res. 49 (2001) 298-307.[31] F.R. Heinzel, V. Bito, L. Biesmans, M. Wu, E. Detre, F. von Wegner, P. Claus, S. Dymarkowski, F. Maes, J. Bogaert, F. Rademakers, J. D'Hooge, K. Sipido, Remodeling of T-tubules and reduced synchrony of Ca2+ release in myocytes from chronically ischemic myocardium, Circ Res. 102 (2008) 338-346.[32] R.C. Balijepalli, A.J. Lokuta, N.A. Maertz, J.M. Buck, R.A. Haworth, H.H. Valdivia, T.J. Kamp, Depletion of T-tubules and specific subcellular changes in sarcolemmal proteins in tachycardia-induced heart failure, Cardiovasc Res. 59 (2003) 67-77.[33] P.S. Haddock, W.A. Coetzee, E. Cho, L. Porter, H. Katoh, D.M. Bers, M.S. Jafri, M. Artman, Subcellular [Ca2+]i gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes, Circ Res. 85 (1999) 415-427.[34] O.J. Kemi, M.A. Hoydal, N. Macquaide, P.M. Haram, L.G. Koch, S.L. Britton, O. Ellingsen, G.L. Smith, U. Wisloff, The effect of exercise training on transverse tubules in normal, remodeled, and reverse remodeled hearts, J Cell Physiol. 226 (2011) 2235-2243.[35] A.R. Lyon, V.O. Nikolaev, M. Miragoli, M.B. Sikkel, H. Paur, L. Benard, J.S. Hulot, E. Kohlbrenner, R.J. Hajjar, N.S. Peters, Y.E. Korchev, K.T. Macleod, S.E. Harding, J. Gorelik, Plasticity of surface structures and beta(2)-adrenergic receptor localization in failing ventricular cardiomyocytes during recovery from heart failure, Circ Heart Fail. 5 (2012) 357-365.[36] R. Wakili, Y.H. Yeh, X. Yan Qi, M. Greiser, D. Chartier, K. Nishida, A. Maguy, L.R. Villeneuve, P. Boknik, N. Voigt, J. Krysiak, S. Kaab, U. Ravens, W.A. Linke, G.J. Stienen, Y. Shi, J.C. Tardif, U. Schotten, D. Dobrev, S. Nattel, Multiple potential molecular contributors to atrial hypocontractility caused by atrial tachycardia remodeling in dogs, Circ Arrhythm Electrophysiol. 3 (2010) 530-541.[37] J.D. Clarke, J.L. Caldwell, M.A. Horn, E.F. Bode, M.A. Richards, M.C. Hall, H.K. Graham, S.J. Briston, D.J. Greensmith, D.A. Eisner, K.M. Dibb, A.W. Trafford, Perturbed atrial calcium handling in an ovine model of heart failure: potential roles for reductions in the L-type calcium current, J Mol Cell Cardiol. 79 (2015) 169-179.[38] A.P. Ziman, N.L. Gomez-Viquez, R.J. Bloch, W.J. Lederer, Excitation-contraction coupling changes during postnatal cardiac development, J Mol Cell Cardiol. 48 (2010) 379-386.[39] S. Seki, M. Nagashima, Y. Yamada, M. Tsutsuura, T. Kobayashi, A. Namiki, N. Tohse, Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes, Cardiovasc Res. 58 (2003) 535-548.[40] F. Sedarat, L. Xu, E.D. Moore, G.F. Tibbits, Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy, Am J Physiol Heart Circ Physiol. 279 (2000) H202-209.[41] G.T. Wetzel, F. Chen, T.S. Klitzner, Ca2+ channel kinetics in acutely isolated fetal, neonatal, and adult rabbit cardiac myocytes, Circulation Research. 72 (1993) 1065-1074.[42] S. Hamaguchi, Y. Kawakami, Y. Honda, K. Nemoto, A. Sano, I. Namekata, H. Tanaka, Developmental changes in excitation-contraction mechanisms of the mouse ventricular

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myocardium as revealed by functional and confocal imaging analyses, J Pharmacol Sci. 123 (2013) 167-175.[43] G.T. Wetzel, F. Chen, T.S. Klitzner, Na+/Ca2+ exchange and cell contraction in isolated neonatal and adult rabbit cardiac myocytes, American Journal of Physiology - Heart and Circulatory Physiology. 268 (1995) H1723-H1733.[44] J. Huang, L. Hove-Madsen, G.F. Tibbits, Ontogeny of Ca2+-induced Ca2+ release in rabbit ventricular myocytes, American Journal of Physiology - Cell Physiology. 294 (2008) C516-C525.[45] P.S. Haddock, W.A. Coetzee, M. Artman, Na+/Ca2+ exchange current and contractions measured under Cl(-)-free conditions in developing rabbit hearts, American Journal of Physiology - Heart and Circulatory Physiology. 273 (1997) H837-H846.*[46] M.L. Munro, C. Soeller, Early transverse tubule development begins in utero in the sheep heart, J Muscle Res Cell Motil. 37 (2017) 195-202.The authors show that, in contrast to small mammals, t-tubules are present at birth in the large mammalian sheep ventricle. This indicates that the onset of t-tubule formation varies between species and may be dependent on developmental maturity at birth.

[47] H.D. Kim, D.J. Kim, I.J. Lee, B.J. Rah, Y. Sawa, J. Schaper, Human fetal heart development after mid-term: morphometry and ultrastructural study, J Mol Cell Cardiol. 24 (1992) 949-965.[48] L.S. Song, E.A. Sobie, S. McCulle, W.J. Lederer, C.W. Balke, H. Cheng, Orphaned ryanodine receptors in the failing heart, Proc Natl Acad Sci U S A. 103 (2006) 4305-4310.[49] M.B. Cannell, D.J. Crossman, C. Soeller, Effect of changes in action potential spike configuration, junctional sarcoplasmic reticulum micro-architecture and altered t-tubule structure in human heart failure, J Muscle Res Cell Motil. 27 (2006) 297-306.**[50] T. Seidel, S. Navankasattusas, A. Ahmad, N.A. Diakos, W.D. Xu, M. Tristani-Firouzi, M.J. Bonios, I. Taleb, D.Y. Li, C.H. Selzman, S.G. Drakos, F.B. Sachse, Sheet-Like Remodeling of the Transverse Tubular System in Human Heart Failure Impairs Excitation-Contraction Coupling and Functional Recovery by Mechanical Unloading, Circulation. 135 (2017) 1632-1645.Here left ventricular biopsies were obtained from patients with chronic heart failure undergoing implantation of a left ventricular assist device (LVAD) to mechanically unload the heart. Biopsies taken during LVAD implantation revealed an increase in the spacing between ryanodine receptors (RyRs) and the cell membrane in chronic heart failure due to transverse (t)-tubule sheet-like remodelling. Mechanical unloading is known to improve cardiac function in some but not all patients. Here a short RyR-membrane distance  was associated with improved ejection fraction following unloading whereas patients with distances over 1um showed no improvement in ejection fraction. This work suggests that an intact t-tubule network may be required for cardiac recovery during unloading.

[51] S. Wei, A. Guo, B. Chen, W. Kutschke, Y.P. Xie, K. Zimmerman, R.M. Weiss, M.E. Anderson, H. Cheng, L.S. Song, T-tubule remodeling during transition from hypertrophy to heart failure, Circ Res. 107 (2010) 520-531.[52] W.E. Louch, H.K. Mork, J. Sexton, T.A. Stromme, P. Laake, I. Sjaastad, O.M. Sejersted, T-tubule disorganization and reduced synchrony of Ca2+ release in murine cardiomyocytes following myocardial infarction, J Physiol. 574 (2006) 519-533.[53] D.J. Crossman, A.A. Young, P.N. Ruygrok, G.P. Nason, D. Baddelely, C. Soeller, M.B. Cannell, T-tubule disease: Relationship between t-tubule organization and regional contractile performance in human dilated cardiomyopathy, J Mol Cell Cardiol. 84 (2015) 170-178.

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[54] L. Sacconi, C. Ferrantini, J. Lotti, R. Coppini, P. Yan, L.M. Loew, C. Tesi, E. Cerbai, C. Poggesi, F.S. Pavone, Action potential propagation in transverse-axial tubular system is impaired in heart failure, Proc Natl Acad Sci U S A. 109 (2012) 5815-5819.[55] A. Yao, Z. Su, A. Nonaka, I. Zubair, K.W. Spitzer, J.H. Bridge, G. Muelheims, J. Ross, Jr., W.H. Barry, Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure, Am J Physiol. 275 (1998) H1441-1448.[56] S.J. Briston, J.L. Caldwell, M.A. Horn, J.D. Clarke, M.A. Richards, D.J. Greensmith, H.K. Graham, M.C. Hall, D.A. Eisner, K.M. Dibb, A.W. Trafford, Impaired beta-adrenergic responsiveness accentuates dysfunctional excitation-contraction coupling in an ovine model of tachypacing-induced heart failure, J Physiol. 589 (2011) 1367-1382.[57] X. Chen, V. Piacentino, 3rd, S. Furukawa, B. Goldman, K.B. Margulies, S.R. Houser, L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices, Circ Res. 91 (2002) 517-524.[58] S. Kettlewell, F.L. Burton, G.L. Smith, A.J. Workman, Chronic myocardial infarction promotes atrial action potential alternans, afterdepolarizations, and fibrillation, Cardiovasc Res. 99 (2013) 215-224.[59] J.L. Caldwell, C.E. Smith, R.F. Taylor, A. Kitmitto, D.A. Eisner, K.M. Dibb, A.W. Trafford, Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1), Circ Res. 115 (2014) 986-996.[60] R.C. Bond, S.M. Bryant, J.J. Watson, J.C. Hancox, C.H. Orchard, A.F. James, Reduced density and altered regulation of rat atrial L-type Ca2+ current in heart failure, Am J Physiol Heart Circ Physiol. 312 (2017) H384-H391.[61] S.N. Hatem, A. Coulombe, E. Balse, Specificities of atrial electrophysiology: Clues to a better understanding of cardiac function and the mechanisms of arrhythmias, J Mol Cell Cardiol. 48 (2010) 90-95.[62] A. Di Maio, K. Karko, R.M. Snopko, R. Mejia-Alvarez, C. Franzini-Armstrong, T-tubule formation in cardiacmyocytes: two possible mechanisms?, J Muscle Res Cell Motil. 28 (2007) 231-241.[63] E. Lee, M. Marcucci, L. Daniell, M. Pypaert, O.A. Weisz, G.C. Ochoa, K. Farsad, M.R. Wenk, P. De Camilli, Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle, Science (New York, N.Y.). 297 (2002) 1193-1196.[64] T.T. Hong, J.W. Smyth, D. Gao, K.Y. Chu, J.M. Vogan, T.S. Fong, B.C. Jensen, H.M. Colecraft, R.M. Shaw, BIN1 localizes the L-type calcium channel to cardiac T-tubules, PLoS Biol. 8 (2010) e1000312.[65] B. Chen, A. Guo, C. Zhang, R. Chen, Y. Zhu, J. Hong, W. Kutschke, K. Zimmerman, R.M. Weiss, L. Zingman, M.E. Anderson, X.H. Wehrens, L.S. Song, Critical roles of junctophilin-2 in T-tubule and excitation-contraction coupling maturation during postnatal development, Cardiovasc Res. 100 (2013) 54-62.[66] J.O. Reynolds, D.Y. Chiang, W. Wang, D.L. Beavers, S.S. Dixit, D.G. Skapura, A.P. Landstrom, L.S. Song, M.J. Ackerman, X.H. Wehrens, Junctophilin-2 is necessary for T-tubule maturation during mouse heart development, Cardiovasc Res. 100 (2013) 44-53.[67] D.R. Scriven, P. Asghari, E.D. Moore, Microarchitecture of the dyad, Cardiovasc Res. 98 (2013) 169-176.**[68] Y. Guo, N.J. VanDusen, L. Zhang, W. Gu, I. Sethi, S. Guatimosim, Q. Ma, B.D. Jardin, Y. Ai, D. Zhang, B. Chen, A. Guo, G.C. Yuan, L.S. Song, W.T. Pu, Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo, Circ Res. 120 (2017) 1874-1888.Using CASAAV (CRISPR/Cas9-AAV9-based somatic mutagenesis) the authors were able to dose-dependently deplete JPH2 demonstrating that JPH2 has only a minor role in t-tubule maturation but is necessary to maintain t-tubule structure in HF. A novel role for RyR in

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regulating t-tubule organisation was also identified using this technique, however the mechanism behind this currently unknown.

[69] T.T. Hong, J.W. Smyth, K.Y. Chu, J.M. Vogan, T.S. Fong, B.C. Jensen, K. Fang, M.K. Halushka, S.D. Russell, H. Colecraft, C.W. Hoopes, K. Ocorr, N.C. Chi, R.M. Shaw, BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes, Heart Rhythm. 9 (2012) 812-820.[70] M. Ibrahim, U. Siedlecka, B. Buyandelger, M. Harada, C. Rao, A. Moshkov, A. Bhargava, M. Schneider, M.H. Yacoub, J. Gorelik, R. Knoll, C.M. Terracciano, A critical role for Telethonin in regulating t-tubule structure and function in the mammalian heart, Hum Mol Genet. 22 (2013) 372-383.[71] Y. Fu, S.A. Shaw, R. Naami, C.L. Vuong, W.A. Basheer, X. Guo, T. Hong, Isoproterenol Promotes Rapid Ryanodine Receptor Movement to Bridging Integrator 1 (BIN1)-Organized Dyads, Circulation. 133 (2016) 388-397.[72] M. Ibrahim, A. Nader, M.H. Yacoub, C. Terracciano, Manipulation of sarcoplasmic reticulum Ca(2+) release in heart failure through mechanical intervention, J Physiol. 593 (2015) 3253-3259.[73] T.T. Hong, R. Cogswell, C.A. James, G. Kang, C.R. Pullinger, M.J. Malloy, J.P. Kane, J. Wojciak, H. Calkins, M.M. Scheinman, Z.H. Tseng, P. Ganz, T. De Marco, D.P. Judge, R.M. Shaw, Plasma BIN1 correlates with heart failure and predicts arrhythmia in patients with arrhythmogenic right ventricular cardiomyopathy, Heart Rhythm. 9 (2012) 961-967.*[74] J.O. Reynolds, A.P. Quick, Q. Wang, D.L. Beavers, L.E. Philippen, J. Showell, G. Barreto-Torres, D.J. Thuerauf, S. Doroudgar, C.C. Glembotski, X.H. Wehrens, Junctophilin-2 gene therapy rescues heart failure by normalizing RyR2-mediated Ca2+ release, Int J Cardiol. 225 (2016) 371-380.This work shows that expression of JPH2 is reduced following transverse aortic constriction. Restoration of JPH2 using gene therapy prevented both deterioration of cardiac function and t-tubule loss in early HF .

[75] A.P. Walden, K.M. Dibb, A.W. Trafford, Differences in intracellular calcium homeostasis between atrial and ventricular myocytes, J Mol Cell Cardiol. 46 (2009) 463-473.

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Table 1. Comparison of key atrial and ventricular features between small and large mammals.

Small mammal ventricle

Large mammal ventricle

Small mammal atria

Large mammal atria

Cell width Median: 24.1 µmRange: 21.3 –

30.2 µm [1,5,16,28,34,75])

Median: 25.8 µm

Range: 23 – 28.7 µm

[1,5,12,31,56],

Median: 13.2 µmRange: 9.5 – 20.7

µm

[6,10,28,58,60,75]

Median: 16.4 µm

Range: 15.2 -22.7 µm

[12-14,37])

T-tubule network characteristics

Well developed with

predominantly transverse elements

[3-5]

Well developed but less

complex in comparison to

small mammalian

ventricle[2-5]

Variable; Most but not all studies

report either complete lack or sparse network of

longitudinally orientated

[6-11]

Similar in structure but less dense than in the

large mammalian

ventricle[7,12-15]

T-tubule density (half distance to t-tubules relative to rat ventricle)

- Fewer tubules

(15% ↑ in distance between

tubules) [59]

Almost completely absent

(441% ↑ in distance between

tubules) [59]

Less dense

(144%↑ in distance between

tubules) [59]

LTCC channel distribution

Concentrated on t-tubules [16,17]

Concentrated on t-tubules

[17]

Distributed evenly across

surface sarcolemma and t-

tubules [6]

Distributed evenly across

surface sarcolemma and t-tubules

[6]

Triggered Ca2+ release

Synchronous Ca2+

release [5,8]Synchronous

Ca2+ release but with some

patchy areas of triggered release

corresponding to regions of

reduced t-tubule density

[5,29]

Cells typically reliant on

propagation for central Ca2+

release. However regions of

triggered Ca2+

release are present on the cell surface

in ‘eager’ sites and along TATs in ‘super hub’

sites [9,10,27,28]

Triggered Ca2+

release in the cell centre in

close proximity to t-

tubules [7,12,14]

Presence of t-tubules at birth

13

[33,38,39] [46,47]Loss of t-tubules in disease (compared to control)

(14.3-54.6% loss)

[48,51,52]

(24.4 - 50%

loss)[30-32,59]

(40-60% loss)

[6,58]

(45-81% loss)

[14,59]

14

Figure captions

Table 1. Comparison of key atrial and ventricular features between small and large mammals. Uniformity and disparity in cell morphology, t-tubule network characteristics and functionality between ventricular and atrial myocytes from small and large mammals. Small mammals include mice, rats and rabbits, with large mammals including sheep, pigs, horses, dogs and humans.

Figure 1. A schematic representation of the t-tubule depicting key excitation-contraction coupling and t-tubule regulatory proteins. T-tubules enable close association between L-type Ca channels (LTCCs) and ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) resulting in uniform Ca2+-induced Ca2+ release throughout the cell. Along with being an important site for Ca2+ entry, Ca2+ extrusion also occurs along the t-tubule via the Na+/Ca2+ exchanger (NCX). T-tubule structure is regulated by a number of proteins including amphiphysin II (AmpII), junctophilin 2 (JPH2) and telethonin (TCAP). AmpII causes membrane curvature to facilitate the formation of t-tubule invaginations. JPH2 enables the formation of dyads between LTCCs and RyRs by regulating t-tubule organisation. TCAP is thought to be involved in the stretch sensitivity of the t-tubule network.

Figure 2. Chamber and species differences in t-tubule density. A, Di-4-ANEPPS stained cell membrane in rat and sheep ventricular myocytes showing an extensive, well ordered t-tubule network across species with some minor variation in density. B, Membrane staining using di-8-ANEPPS or di-4-ANEPPS in rat and sheep atrial myocytes to show species-related differences in t-tubule organisation and density. i-ii, Rat atrial cells typically lack t-tubules or contain sparse longitudinally orientated transverse-axial tubules (TATs) that are structurally different to t-tubules observed in sheep atrial myocytes. iii, In large mammalian atrial cells t-tubules are mainly transverse in orientation, similar to those observed in the ventricle but at a lower density. Panel Bii is modified from Kirk et al.[10]. Scale bars denote 10µm.

Figure 3. T-tubule growth during neonatal development and loss in heart failure.A, Di-8-ANEPPS stained ventricular myocytes from 10 day, 15 day, 20 day old and adult rats showing an absence of t-tubules 10 days post birth and their progressive postnatal development into adulthood. B, Di-4-ANEPPS stained ventricular and atrial myocytes from control (CON) and heart failure (HF) sheep showing the loss of t-tubules in heart failure. Panel A is modified from Ziman et al. [38]. Scale bars denote 10µm.

15