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REVIEW

Resurgence of Cardiac T-Tubule Research

Fabien Brette¹ and Clive Orchard²

¹ Faculty of Life Sciences, University of Manchester, Manchester; and
² Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom f.brette{at}manchester.ac.uk

	Abstract

 
The transverse tubules of mammalian cardiac ventricular myocytes are invaginations of the surface membrane. Recent data have revealed that their structure and function are more complex than previously believed. Here, we review current knowledge about their role in cardiac function, focusing on Ca²⁺ signaling and changes observed in pathological conditions.

	Introduction

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
Every heartbeat is triggered by the propagation of an action potential through the heart. During the cardiac action potential, Ca²⁺ enters each cardiac myocyte through L-type Ca²⁺ channels [as the Ca²⁺ current (I_Ca)] and activates adjacent Ca²⁺ release channels [ryanodine receptors (RyR)] in the membrane of the intra-cellular Ca²⁺ store, the sarcoplasmic reticulum (SR), causing Ca²⁺ release from the SR (5). Ventricular myocytes, which cause contraction of the ventricles of the heart, have invaginations of the surface membrane, called transverse (t-) tubules, which are important in ensuring rapid and synchronous contraction of the cell, and hence of the heart (13). This brief review will describe recent evidence that t-tubules are regions of the cell membrane that play a specialized role in transmembrane ion flux, and hence cell function, and that this role may change, and hence contribute to the altered function observed, during heart failure.

	Structure of Cardiac T-Tubules

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
Mammalian cardiac myocytes differ from those of fish, amphibians, reptiles, and birds by having an extensive t-tubule system (4, 9, 34, 67). Within mammalian cardiac tissue, t-tubules occur predominantly in ventricular myocytes, being absent from pacemaking and conducting tissue and from most atrial myocytes (1, 24), although some may show a sparse distribution (e.g., Ref. 46). In ventricular myocytes, t-tubules are labile: they are absent in neonatal cells (37), and decrease when cells are kept in culture (51, 53), but little is known about the mechanisms underlying t-tubule expression and maintenance. However, in CHO cells, expression of amphisin-2, a protein that can link the plasma membrane and submembranous cytosolic scaffolds, generates narrow tubules that are continuous with the plasma membrane (49). T-tubule development also shows similarities to the development of caveolae, which requires cholesterol and caveolin-3 (21, 31, 41). Interestingly, caveolin-3-null mice show abnormalities in t-tubule organization in skeletal muscle (33), although it remains to be shown whether similar changes occur in cardiac myocytes. Cardiac t-tubule diameter is typically 100–300 nm (69), which is much larger than t-tubules in skeletal muscle [20–40 nm (32, 63)]. T-tubules are found at ~1.8-µm intervals along the long axis of the cell; roughly at every Z line. Along a Z line, t-tubules occur at regular intervals of ~1.2 µm (45) and run deep into the ventricular cell. Although most t-tubules are organized radially, they also branch and have longitudinal extensions (29, 55, 69), which can comprise up to 40% of the total t-tubule volume (69). Thus t-tubules form a complex network of branching tubules with both transverse and longitudinal elements rather than a simple transverse system of tubules, as its name might suggest. Alternative names have therefore been proposed, such as the transverse-axial tubular system (TATS), sarcolemmal Z rete, or sarcolemmal tubule network (4, 29, 69), although t-tubules remains the standard term. This network of membrane within the cell conducts the cardiac action potential into the center of the myocyte.

	T-Tubules and Electrical Activity

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
Physical and functional distribution of ion channels and transporters
The function of t-tubules depends not only on their structure but also on the proteins within and adjacent to the t-tubule membrane. Immunocytochemistry in conjunction with confocal microscopy has been widely used to investigate the location of proteins within cardiac ventricular myocytes. Many important proteins are present in the t-tubule membrane, including ion channels such as L-type Ca²⁺ channels (LTCC), Na⁺ channels, and K⁺ channels; and transporters such as Na⁺/Ca²⁺ exchanger (NCX) and Na⁺/K⁺ ATPase. Key proteins involved in the modulation of cardiac function are also present at the t-tubules, including nitric oxide synthase type 3 and cAMP-dependent pathway proteins, such as the ß-adrenoceptor, G_s, adenylate cyclase, and AKAP (see Ref. 13 for review). In addition, LTCCs are highly co-localized with RyRs at the dyad, where the sarcolemmal and junctional SR membranes are closely apposed (20, 65); dyads are more abundant at the t-tubules than at the surface membrane, e.g., 25% at the cell surface compared with 75% at the t-tubules in rat (18). Moreover, the SR Ca²⁺ ATPase (SERCA2) and its regulatory protein phospholamban are located throughout the SR membrane including at the Z line, adjacent to the t-tubule (16, 54), although the location of SERCA2 relative to the dyad is not yet known.

Although immunocytochemistry has been useful to localize proteins in cardiac myocytes, protein distribution is not necessarily reflected in protein function, which also depends on local environment and regulation and accessory proteins (13). Furthermore, quantification of protein densities from immunocytochemistry is difficult and may depend on the antibodies used. To quantify protein function in the t-tubules, a different approach is required. The first studies to show that membrane currents are located predominantly in the t-tubules used the fact that such channels are in a restricted diffusion space. Thus, when the composition of the solution bathing a cell was changed rapidly, some currents, for example I_Ca, showed an initial rapid change followed by a slower change. The first phase represented about 36% of I_Ca, the slow phase 64%, suggesting that these fractions of I_Ca are located in the surface and t-tubule membranes, respectively (66). More recently, a different approach has been developed: the detubulation technique. This is an adaptation of the "osmotic shock" technique used previously to detubulate skeletal muscle (e.g., Ref. 43). The osmotic shock and consequent changes of cell volume, caused by application and removal of a membrane permeant agent (formamide), result in the t-tubules being physically and functionally uncoupled from the surface membrane (44). Importantly, this procedure has no effect on the function of rat atrial myocytes, which lack t-tubules (11), which strongly argues against a direct effect of formamide. Detubulated rat ventricular myocytes show a decrease in cell capacitance (which reflects membrane area) of ~30% (14–18, 44, 47, 75), a value close to the estimate of membrane present within the t-tubules obtained by electron microscopy (56). This technique enables functional mapping of currents mediated by ion channels and transporters. Using detubulation, we have shown that 80% of I_Ca (17) and 63% of I_NCX (25) are present in the t-tubules of rat ventricular myocytes. Thus the major Ca²⁺ flux pathways are more concentrated in the t-tubules than in the surface membrane (Table 1). Some Na⁺ handling proteins are also concentrated in the t-tubules, e.g., Na⁺-K⁺-ATPase and the neuronal isoform of the Na⁺ current (14, 15, 25). In contrast, K⁺ currents are evenly distributed between the surface and t-tubule membranes, except for I_SS which is concentrated at the t-tubules (47). Interestingly, we have found only one ionic current, which is more concentrated at the surface membrane than the t-tubules: the cardiac isoform of Na⁺ current (14, 15). This technique has been adopted by other laboratories, which have shown similar results for the distribution of NCX (72) and have revealed the isoform distribution of Na⁺-K⁺-ATPase (3).

Investigation of the electrical properties of the t-tubules is also hampered by our inability to record the electrical activity of the t-tubule membrane only. We have, however, investigated the characteristics of the action potential in control (t-tubules and surface membrane) and detubulated (surface membrane only) myocytes to obtain an indirect estimate of the electrical activity of the t-tubules. The main effect of loss of t-tubules was to decrease action potential duration, whereas action potential amplitude and resting membrane potential were unchanged (18). It is likely that the decrease in action potential duration is due to loss of I_Ca and I_NCX, which carry positive charge into the cell and are concentrated at the t-tubules, with a limited role for K⁺ currents, which are evenly distributed (Table 1). Calculations from these data suggest that the t-tubule action potential should be ~5.5 longer than at the cell surface. This challenging speculation of course requires experimental confirmation when direct recording from t-tubule membrane becomes possible. However, in a normal myocyte, the action potential will be uniform throughout the cell membrane (the mean of that generated by the two membranes) because of the tight electrical coupling of the surface and t-tubule membranes. This has been described recently using mathematical modeling (57); indeed, the action potential is delayed by only 2–3 ms in the t-tubules, as previously described in skeletal myocytes (42).

	T-Tubules and Calcium Cycling

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
It was assumed for many years that t-tubules were simple invaginations of the surface membrane of cardiac ventricular myocytes, which allowed propagation of the action potential to the cell interior, allowing spatially and temporally synchronous Ca²⁺ release throughout the cell. This idea received support with the development of Ca²⁺-sensitive dyes and fast imaging techniques, which showed that cells lacking t-tubules, for example those from the atria and Purkinje fibers (8, 24, 40), cultured ventricular cells (51), and cells from the ventricles of newborn animals (37), showed asynchronous Ca²⁺ release, with Ca²⁺ rising initially at the surface membrane, in contrast to ventricular myocytes that showed synchronous Ca²⁺ release (11, 16, 37, 75). However, it now appears that t-tubules are not simple invaginations; many of the key proteins involved in excitation-contraction coupling are located predominantly at the t-tubules (Table 1), which suggests that they play a specialized and important role in Ca²⁺ handling and excitation-contraction coupling.

	The T-Tubules Represent a Specialized Region for Transmembrane Ion Flux

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
The detubulation technique has allowed quantification of the distribution of membrane currents between the t-tubule and surface membranes (above). It is striking that the currents involved in Ca²⁺ handling appear to be located predominantly within the t-tubules (Table 1). This is interesting for a number of reasons, not least because it places them within a domain with restricted diffusion access to the bulk extracellular space. An intracellular restricted diffusion space appears to exist under the sarcolemma ["fuzzy space" (48)] so that the concentration of ions adjacent to the sarcolemma, and thus to the intracellular face of ion flux pathways, may be different from that in the bulk intracellular space (4). It now seems feasible that concentration of ion flux pathways in the t-tubules, coupled to restricted diffusion, may result in these proteins also being exposed to an extracellular ion concentration that is different from that in the bulk extracellular solution. Computer modelling suggests that this may result in Ca²⁺ depletion in the t-tubule lumen during activity, which may limit Ca²⁺ influx (57). Similarly, a recent study has shown slow K⁺ diffusion in the t-tubules (~85 µm²/s), which can lead to K⁺ accumulation (71). This might be particularly important in modulating cell function in small rodents (mouse and rat), in which repolarization is due predominantly to the large transient outward current.

	Ca2+ Influx and Ca2+ Release at the T-Tubules

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
Ca²⁺ influx via the LTCCs triggers Ca²⁺ release from the SR; it is predominantly this Ca²⁺ that activates the contractile proteins and causes contraction of the cardiac ventricular myocyte. I_Ca also helps to load the SR with Ca²⁺ ready for subsequent release (27) and thus plays a pivotal role in cardiac cell function.

Comparison of I_Ca in intact ventricular myocytes, in which it flows predominantly across the t-tubule membrane, with that in detubulated myocytes, in which it flows across the surface membrane, suggests that the current is different at the two sites. Detubulation causes not just a marked decrease in current amplitude, consistent with loss of channels (above), but the remaining current exhibits a slow time course of inactivation (17). This implies that t-tubular I_Ca is large but inactivates quickly, whereas that at the surface membrane is small but inactivates slowly. The rapid inactivation of t-tubular I_Ca does not appear to be due to depletion of Ca²⁺ within the t-tubule lumen or to a difference in voltage-dependent inactivation, because inactivation of I_Ca is the same in control and detubulated myocytes when Ba²⁺ is used as the charge carrier (17). However inhibition of SR Ca²⁺ release abolishes the difference in inactivation rate, suggesting that inactivation of I_Ca by Ca²⁺ released from the SR is more profound at the t-tubules than at the surface membrane. Although the mechanism of this differential Ca²⁺-dependent inactivation is unknown, it does not appear to be due to differences in SR Ca²⁺ release at the two sites (below).

The difference in the kinetics of I_Ca at the t-tubules and surface membrane is important, because it means that the amount of Ca²⁺ entering the cell via I_Ca at the surface membrane is larger than might be expected from its amplitude. Since the inactivation phase of I_Ca appears to be important in loading the SR with Ca²⁺, this suggests that surface I_Ca may be "specialized" for this role, an idea supported by the observation that, although detubulation slows the rate of SR loading, the slowing is not as great as might be expected from the decrease in current amplitude (10). Conversely, a large, rapidly inactivating I_Ca, as in the t-tubules, has the characteristics required for an effective trigger for Ca²⁺ release, which depends more of the early phase of I_Ca (27, 60).

Although the characteristics of I_Ca appear to be different at the t-tubule and surface membranes, the coupling of I_Ca to SR Ca²⁺ release is surprisingly consistent: from the amplitude of I_Ca in control and detubulated myocytes, and using reasonable assumptions about single channel current, it is possible to calculate the number of Ca channels in the t-tubule and surface membranes of a rat ventricular myocyte (143,400 and 48,200, respectively). Using electron microscopy data to calculate the number of RyR clusters (Ca²⁺ release sites or junctions) at the t-tubules and cell surface, it is thus possible to calculate the number of Ca²⁺ channels per junction: ~35 at the two sites (18) (FIGURE 1). This is borne out in measurements of the amount of Ca²⁺ released for a given Ca²⁺ influx at the two sites, which is not significantly different (17).

View larger version (100K): [in this window] [in a new window]   FIGURE 1. Proteins involved in calcium signaling in mammalian ventricular myocytes Junctional sarcoplasmic reticulum (SR) membrane in close proximity to the sarcolemma forms dyads, which are present mainly in the transverse tubules (TT) but also at the surface membrane. Dyads comprise a cluster of ryanodine receptors (RyRs) adjacent to, and under the functional control of, L-type Ca2+ channels; this forms a Ca2+ release unit. Proteins involved in Ca2+ re-uptake (SR Ca2+-ATPase, SERCA2) and extrusion [Na+/Ca2+ exchanger (NCX)] are preferentially located within or near the transverse tubules.

Local regulation of Ca²⁺ flux at the t-tubules
Local regulation of membrane proteins is known to occur in cardiac myocytes. ß₂-Adrenergic stimulation, for example, stimulates I_Ca without affecting other proteins, apparently because phosphodiesterases spatially limit the effect of locally produced cAMP (28). Caveolae [50- to 80-nm diameter invaginations of the surface and t-tubule membranes (4)] have also been implicated in compartmentalized signaling in cardiac myocytes (see Ref. 23 for review). Immunocytochemistry has shown that many key components of major signalling pathways are located predominantly at the t-tubules, and some proteins, such as RyR, form macromolecular complexes that include kinases and phosphatases whose action is therefore localized (6, 74). It has also been shown that I_Ca in the t-tubules is inactivated more rapidly by Ca²⁺ released from the SR, compared with I_Ca at the surface membrane, even though the amount of Ca²⁺ released by a given Ca²⁺ influx is the same at the two sites (above). This inactivation depends on calmodulin, which is prebound to the channel (26, 76); the data suggest, therefore, that this Ca²⁺ activated pathway is more active at the t-tubules than the surface membrane, although the reason is unknown. Similarly, it has been shown that the fractional increase of I_Ca produced by the adrenergic agonist isoprenaline is greater in control than in detubulated myocytes, suggesting that I_Ca is better coupled to the adrenergic pathway at the t-tubules than at the surface membrane (16). In contrast, isoprenaline-induced phosphorylation of the regulatory protein phospholamban, which is found in the SR membrane throughout the cell, is unaffected by detubulation (16).

These data provide evidence that ion flux pathways are differentially regulated at the t-tubules and surface membrane; as a consequence, their function may be different at the two sites, particularly in the presence of tonic activation of signalling pathways.

	T-Tubules and Pathophysiology

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
Surprisingly few studies have examined t-tubule structure in living cells from heart failure models or patients. In a canine model of tachycardia-induced heart failure, a marked loss of t-tubules in ventricular myocytes has been described (2, 39). Loss of t-tubules has also been observed in a rabbit model of heart failure (t-tubule area 59% of sham) (59). In human failing myocytes, the issue of whether t-tubules are altered is starting to be addressed. Although a prominent t-tubule network has been reported in failing human ventricular myocytes, this study was conducted without control myocytes (51). Another recent study reported an increase in the size of the t-tubules and more longitudinal extensions than in control human heart (19).

Although loss of t-tubules cannot be responsible for all the changes of Ca²⁺ handling that occur during heart failure, it is notable that detubulation results in many of the functional changes observed in heart failure, suggesting that loss of t-tubules in heart failure may contribute to and exacerbate the phenotype observed in failure. These changes include a reduced and slowed Ca²⁺ transient, less synchronized SR Ca²⁺ release, a negative force-frequency relationship, a decreased response of I_Ca to ß-adrenergic stimulation, a decreased contractile response to cardiac glycosides, and decreased frequency-dependent facilitation of I_Ca (10, 16, 17, 30).

However, it remains unclear how t-tubule disorganization influences excitation-contraction coupling during heart failure. Several recent studies have correlated t-tubule remodelling and desynchronization of SR Ca²⁺ release (19, 51, 52, 70). Instead of marked t-tubule loss (above), two recent studies showed subtle reorganization of the t-tubular network (52, 70) that, in these animal models of heart failure (mouse and rat), was directly linked to changes in the synchronicity Ca²⁺ release. Changes in t-tubule organization may alter the spatial distribution of LTCCs (which are concentrated at the t-tubules, above), whereas RyR distribution remains regular, resulting in some orphaned RyRs (70). Orphaned RyRs (70) and/or an increased number of longitudinal t-tubules (52) are likely to induce a delay in SR Ca²⁺ release because of the increased average distance between LTCCs and RyRs, thereby causing desynchronization of SR Ca²⁺ release (51, 52, 70). The same subtle t-tubule reorganization has been observed in failing human heart (19), and computer modelling has shown that this can reduce the synchrony of Ca²⁺ spark production and lead to the appearance of late Ca²⁺ sparks and greater nonuniformity of intracellular Ca²⁺ (19), as observed in animal models of heart failure (50, 58) (FIGURE 2). These data suggest that geometric factors can play an important role in the pathophysiology of human heart failure. This is consistent with the observation of decreased excitation-contraction coupling gain (i.e., the ability of a given I_Ca to trigger SR Ca²⁺ release) in a rat model of heart failure in which it was speculated that altered excitation-contraction coupling was due to t-tubule remodelling (35, 36).

View larger version (24K): [in this window] [in a new window]   FIGURE 2. T-tubules and calcium signaling in mammalian ventricular myocytes Each panel shows a cartoon of the cell structure (top), a confocal linescan image of intracellular Ca2+ (middle), and the Ca2+ transient (bottom). In a normal ventricular myocyte (left), there is an extensive t-tubule network that ensures synchronization of calcium release. In a myocyte without t-tubules (i.e., detubulated, atrial, neonatal; middle), Ca2+ release is initiated at the edge of the cell and then propagates toward the center. In a myocyte where there is a subtle reorganization of the t-tubules (e.g., some animal models and human myocytes from failing hearts; right), desynchronization of the Ca2+ transient is observed.

	Conclusions and Future Directions

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 
The last few years have seen an enormous increase in our knowledge and understanding of cardiac t-tubules. However, it is frustrating that there is still no method that allows direct recording from the t-tubule membrane, although voltage-sensitive dyes and the ability to isolate a single t-tubule using scanning ion conductance microscopy hold promise for the future. Until then, our knowledge is indirect: we still have, for example, no direct measurements of currents flowing across the t-tubule membrane or knowledge of the environment in which the ion flux proteins are located, for example, the composition of the t-tubule membrane. More fundamentally, it is still not clear how t-tubules, which appear to be extremely labile, are formed and maintained, or how ion flux pathways are localized to the t-tubule membrane, or how t-tubule structure, composition, and function change during cardiac pathology. Until we are able to make such measurements and answer these questions, our knowledge of the importance of the t-tubules, so tantilizingly revealed in the last few years, will remain hazy.

	Acknowledgments

 
We thank Dr. Laurent Sallé for making a preliminary version of FIGURE 1.

Work in our laboratories is supported by the Wellcome Trust and British Heart Foundation. F. Brette is a Wellcome Trust Research Fellow.

	References

Top Introduction Structure of Cardiac T-Tubules T-Tubules and Electrical... T-Tubules and Calcium Cycling The T-Tubules Represent a... Ca2+ Influx and Ca2+... T-Tubules and Pathophysiology Conclusions and Future... References

 

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