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t-tubules and sarcoplasmic reticulum function in cardiac ventricular myocytes

Clive Orchard, Fabien Brette
DOI: http://dx.doi.org/10.1093/cvr/cvm002 237-244 First published online: 1 January 2007

Abstract

Although the existence of t-tubules in mammalian cardiac ventricular myocytes has been recognized for a long time, it now appears that their structure and function are more complex than previously believed. Recent work has provided evidence that many of the key proteins underlying excitation–contraction coupling are located predominantly at the t-tubules. L-type Ca2+ current (ICa) flowing across the t-tubule membrane provides a rapidly inactivating Ca2+ influx that triggers Ca2+ release from the sarcoplasmic reticulum (SR), thereby allowing rapid and synchronous Ca2+ release throughout the cell; ICa at the t-tubules also appears to be more sensitive than that at the surface membrane to regulation by beta-adrenergic stimulation and intracellular Ca2+. In contrast, although its density is lower, ICa flowing across the surface membrane inactivates slowly, and thus may help load the SR with Ca2+. There is also increasing evidence that many of the mechanisms that remove Ca2+ from the cytoplasm are located predominantly at the t-tubules, which therefore play an important role in determining cellular, and hence SR, Ca2+ content. Thus, the t-tubules appear to play a central role in the increase and subsequent decrease of Ca2+ during the systolic Ca2+ transient. Remodelling of the t-tubules has been reported in cardiac pathologies, and may play a role in the altered cellular, and hence cardiac, function observed in such conditions.

Keywords
  • Cardiac muscle
  • t-tubules
  • Calcium
  • Excitation–contraction coupling
  • Sarcoplasmic reticulum
  • Heart failure

1. Introduction

During the cardiac action potential, Ca2+ influx across the cell membrane via L-type Ca2+ channels (LTCCs) triggers the release of more Ca2+ from the sarcoplasmic reticulum (SR) by activating ryanodine receptors (RyRs) in the adjacent SR membrane.1 The rise of intracellular Ca2+ that activates the contractile proteins, the systolic Ca2+ transient, is the spatial and temporal sum of such local releases.2 The fraction of the SR Ca2+ content that is released depends on SR Ca2+ content and the size of the Ca2+ trigger.3 The fraction of the Ca2+ transient due to SR Ca2+ release depends on species, age, pathology, and conditions, but estimates in mammals vary from 77% in rabbit to 92% in rat.4

Relaxation is brought about by removal of Ca2+ from the cell cytoplasm by two main routes: the SR Ca2+ ATPase (SERCA), which is regulated by phospholamban (PLB), pumps Ca2+ back into the SR, while Na+/Ca2+ exchange (NCX) uses the inwardly directed electrochemical gradient for Na+ to extrude Ca2+ from the cell.1 The fraction of Ca2+ removed from the cytoplasm by each of these pathways depends on their relative activities, although in the steady state the amount of Ca2+ extruded from the cell must equal that entering during each beat.5

Ca2+ flux via each of these pathways is regulated by second messenger pathways: LTCC, RyR, SERCA, and PLB have all been reported to be phosphorylated by protein kinase A (PKA) and Ca-calmodulin dependent protein kinase II (CaMKII), and NCX by PKA. Phosphorylation has been reported to, respectively: (i) increase L-type Ca2+ current (ICa), although calmodulin (CaM) and CaMKII-induced phosphorylation also play a role in Ca2+ dependent inactivation of ICa;6 (ii) increase the open probability of RyR and its sensitivity to trigger Ca2+;7 it has also been suggested that hyper-phosphorylation of RyR during heart failure increases Ca2+ leak,8 although this has proved controversial;9 (iii) increase SERCA activity,10 although recent work has failed to find significant phosphorylation, and it currently seems unlikely that such phosphorylation plays a significant role in vivo;11 (iv) cause PLB to detach from SERCA, increasing pump activity, and hence SR Ca2+ content;12 (v) increase NCX activity,13,14 although some studies have shown little effect of phosphorylation on NCX.15,16

We have recently developed a technique to physically and functionally uncouple the t-tubules from the surface membrane of cardiac myocytes (detubulation) using osmotic shock induced by the rapid application and removal of the membrane-permeant agent formamide.17,18 In conjunction with other approaches, this has shown that key proteins involved in excitation–contraction coupling, as well as many of the proteins involved in the regulatory second messenger pathways, are located predominantly at the t-tubules.19 These tubules are invaginations of the surface membrane that occur at each Z-line, but branch within the cell to form a complex network with both transverse and longitudinal elements.20 This arrangement ensures that Ca2+ release occurs synchronously throughout the cell, and that Ca2+ can be extruded rapidly, since no part of the cytoplasm is more than ∼1 µm from the nearest t-tubule and its Ca2+ flux pathways. Interestingly, t-tubule density appears to be lower in large mammals (e.g. pig) than in small mammals, such as mouse,21 which have higher heart rates; this is consistent with the idea that they help determine the rate of increase and decrease of cytoplasmic Ca2+, which may need to occur faster in species with higher heart rates.

Thus, the SR plays a key role in cardiac excitation–contraction coupling, and its function is inextricably linked to the structure and function of the adjacent t-tubule system, which contains many of the proteins that determine SR function. In this review, we will explore the role of the t-tubules in determining SR function, and how this might change in pathological conditions.

2. Ca2+ Release from sarcoplasmic reticulum

2.1 Triggered sarcoplasmic reticulum Ca2+ release

Ca2+ release from the SR is triggered by Ca2+ influx across the cell membrane. ICa flowing through LTCCs is the primary source of trigger Ca2+. T-type Ca2+ current and NCX can trigger Ca2+ release from the SR, but they normally play a minor role;22,23 this may be because these proteins are poorly expressed, are located distant from RyRs,24 or because the relatively low rate of Ca2+ influx via these routes constitutes a poor trigger.25 Immunohistological and functional studies have shown that LTCCs are located predominantly in the t-tubules (reviewed in Brette and Orchard),19 in close proximity to RyRs24 at the dyad: the junctional region (couplon) where Ca2+ influx triggers SR Ca2+ release.

Investigation of ICa within the t-tubules requires good voltage clamp control of the t-tubule membrane. Poor electrical coupling between the t-tubule and surface membranes, or propagated responses along the t-tubule membrane, could result in voltage escape, and thus erroneous current–voltage relations. However, at least two pieces of experimental evidence suggest good voltage control of the t-tubule membrane: first, voltage clamp pulses result in a single exponential decay of whole cell capacitance current,26 consistent with tight electrical coupling between the surface and t-tubule membranes; secondly, time to peak INa is unaltered at different potentials following detubulation, and little affected by differences in series resistance over the range 1–4 MΩ in control and detubulated myocytes.27 The lack of effect of small changes of series resistance and, importantly, the observation that detubulation did not alter INa, which suggests that there were no errors arising from the presence of the t-tubules, suggest that the t-tubules are adequately voltage clamped. These data suggest that the membrane potential is likely to be uniform during all but the largest and fastest changes of membrane voltage induced either experimentally using voltage clamp, or during physiological activation of membrane currents, suggesting that ICa within the t-tubules can be measured accurately. We have, therefore, used the decrease in ICa following detubulation to calculate that ∼75–80% of ICa flows across the t-tubule membrane in a rat myocyte. The dimensions of these cells are ∼100 × 20 × 20 µm; assuming that the specific capacitance of the surface membrane is 1 µF/cm2, they have a total surface area of 18 600 µm2, with 48 200 Ca2+ channels at the cell surface and 143 400 in the t-tubules, giving densities of 3.8 and 24.3 Ca2+ channels/μm2, respectively.28

In mammalian myocytes, there appear to be 4–10 RyR per Ca2+ channel at each dyad, with the Ca2+ channels situated apparently randomly with respect to RyR within the junction.4 Interestingly, these proteins appear to be located predominantly in the transverse elements of the t-tubule system,29 suggesting inhomogeneity even within the t-tubules. It has been suggested, however, that the size of the junction may differ in different regions of the cell.30 To investigate whether this difference is between the t-tubule and surface membranes, we used EM data of junctional areas31 and our measurements of Ca2+ channel numbers to calculate that a typical junction in a rat ventricular myocyte contains ∼35 Ca2+ channels at both the cell surface and t-tubules.28 This is somewhat higher than other reports (10–25 Ca2+ channels),4 although rat ventricular myocytes have more RyRs per junction than other species,31 so that the RyR:LTCC ratio of ∼7 is similar to other species.4 The constancy of the RyR:LTCC ratio32,33 suggests that mechanisms exist to determine co-localization of LTCCs and RyRs.

Consistent with the suggestion that junctions at the cell surface and t-tubular membranes contain the same number of Ca2+ channels, and that the RyR:Ca2+ channel ratio is constant, the gain of SR Ca2+ release (Ca2+ release for a given Ca2+ trigger) is not significantly different at the surface and t-tubule membranes.34 However, since the majority of Ca2+ release sites are located at the t-tubules, they appear to be the major site for Ca2+ entry and release, and hence for excitation–contraction coupling: Ca2+ sparks occur predominantly near the t-tubules35 and localized Ca2+ release (Ca2+ spikes) occurs at discrete sites at the Z-line.36 The importance of the Ca2+ release occurring at the t-tubules has been demonstrated in cells lacking t-tubules in which the electrically stimulated rise of intracellular Ca2+ initially occurs close to the cell membrane and then either diffuses (Purkinje and neonatal cells)37,38 or propagates via Ca2+-induced Ca2+ release (atrial, cultured, and detubulated cells)3941 into the cell.

Fabiato showed that the effectiveness of Ca2+ as a trigger for SR Ca2+ release depends on the size and the rate of change of Ca2+ at the RyR.25 It has since become clear that the large and rapid Ca2+ influx occurring during the first 5–10 ms of ICa is responsible for triggering SR Ca2+ release;42 the latter part of ICa appears to load the SR with Ca2+ that is released in the subsequent contraction.43 Inactivation of ICa is significantly slower in detubulated cells (i.e. at the surface membrane) than in control cells, but this difference is abolished by ryanodine or BAPTA;34 this suggests that Ca2+ released from the SR inactivates ICa to a greater extent in the t-tubules than at the surface membrane. This does not appear to be due to: (i) differences in local SR Ca2+ release, because the gain of Ca2+ release is the same at the two sites; (ii) Ca2+ depletion within the t-tubules or differences in voltage dependent inactivation, because the difference was abolished when Ba2+ was used as the charge carrier; (iii) smaller Ca2+ influx, because inactivation was little affected by ryanodine in detubulated cells, and was the same in control and detubulated cells in the presence of ryanodine, despite differences in the amplitude of ICa. These data have three important implications: first, that ICa in the t-tubules is large, but inactivates rapidly due to marked Ca2+ dependent inactivation; this gives it the characteristics required for a Ca2+ trigger. Secondly, that the more slowly inactivating ICa at the surface membrane may be important in loading the SR with Ca2+; this will be discussed below. Third, that regulation of ICa by Ca2+ is more marked at the t-tubules; this implies that ICa is differentially modulated at the surface and t-tubule membranes, and that the t-tubules are a key site for the regulation of trans-sarcolemmal Ca2+ flux by Ca2+ released from the SR.

Local regulation of ICa has been suggested previously, although not differential regulation between the t-tubule and surface membranes. The observation of marked regulation by SR Ca2+ release means that the t-tubules may play an important role in cellular Ca2+ homeostasis: an increase in SR Ca2+ release decreases Ca2+ influx via ICa and increases efflux via NCX,4446 thus restoring Ca2+ balance (autoregulation).47 If ICa in the t-tubules is inactivated to a greater extent by a given SR Ca2+ release than ICa at the surface membrane, and Ca2+ release occurs predominantly at the t-tubules, it appears likely that much of this autoregulation occurs at the t-tubules. NCX appears to have privileged access to Ca2+ released from the SR,48 and the majority of NCX function appears to be located in the t-tubules,41,49 so that the effect of changes in SR Ca2+ release on Ca2+ extrusion by NCX may also be most marked in the t-tubule membrane.

ICa may be modulated by other factors, in addition to Ca2+ in the dyadic cleft. First, since the t-tubules represent a space with restricted diffusion and a high concentration of Ca2+ transport proteins, changes of Ca2+ may occur within the t-tubule lumen, which would modulate membrane currents; in this case, the extracellular face of the protein would be exposed to a Ca2+ concentration different from the bulk extracellular solution, in the same way that the intracellular face is exposed to a concentration in the fuzzy space that is different from that in the bulk intracellular compartment. When Ba2+ is used as the charge carrier, the rate of inactivation of current through the LTCC is the same in the surface and t-tubule membranes, suggesting that depletion does not contribute to inactivation.34 However, these experiments were performed at a low stimulation rate to allow recovery from inactivation between pulses. Simulation of ion concentration changes within the t-tubule lumen using biophysically realistic computer models of the cardiac ventricular myocyte incorporating a t-tubule system and known distributions of Ca2+ flux pathways and diffusion rates suggest that at physiological stimulation rates Ca2+ depletion may occur in the t-tubule lumen, and that this decreases ICa and the amplitude of the Ca2+ transient as a result of the decrease in both the trigger and loading functions of ICa.50 The rapid inactivation of ICa in the t-tubules may help limit such depletion. Secondly, stimulation of ICa by PKA-induced phosphorylation may be different at the surface and t-tubule membranes. Many of the key proteins are located predominantly at the t-tubules and localized stimulation of ICa by beta-adrenergic stimulation has been reported previously.51 More recently, we have shown that the beta-adrenergic agonist isoprenaline has a greater stimulatory effect on ICa in intact than in detubulated myocytes, suggesting that the beta-adrenergic system is better coupled to the Ca2+ channel in the t-tubules than at the surface membrane.52

Interestingly, preliminary data suggest that phosphorylation of RyR following application of isoprenaline is also inhibited by loss of the t-tubules (Brette, Rodriguez, Colyer and Orchard, unpublished results), suggesting that local signalling may underlie RyR phosphorylation. Although not surprising in one way, because RyR forms part of a macromolecular complex that includes kinases and phosphatases,7 it is surprising in another, because in detubulated myocytes the speed of propagation of Ca2+ from the cell surface is increased by isoprenaline.53 This is due in part to an increase in SR Ca2+ load, which increases the rate of propagation, and an obvious explanation is that RyR phosphorylation also contributes to the increased propagation velocity. The explanation for this discrepancy remains to be elucidated.

In addition to changes of Ca2+ influx brought about by direct regulation of ICa, Na+ and K+ channels, by altering action potential configuration, may also alter Ca2+ fluxes. Recent work has shown that the neuronal Na+ current isoform is located predominantly in the t-tubules whereas the cardiac form is found predominantly at the surface membrane;27,54 the functional consequence of these isoform distributions is unclear, particularly since the distribution of total (cardiac + neuronal) INa is uniform. In contrast, K+ currents are evenly distributed between the surface and t-tubule membranes, except for ISS, which is concentrated at the t-tubules.55 The net effect of detubulation is to decrease action potential duration, with no effect on action potential amplitude or resting potential,28 presumably due to loss of ICa and INCX. Although this suggests that the action potential may be longer in the t-tubules than at the surface membrane, the action potential in the intact cell will be uniform throughout the cell membrane—the mean of that generated by the two membranes, because of their tight electrical coupling.

2.2 Spontaneous sarcoplasmic reticulum Ca2+ release

Spontaneous Ca2+ releases from the SR (Ca2+ sparks) occur as a result of spontaneous RyR openings.56 Ca2+ sparks occur predominantly at the Z-lines of cardiac ventricular myocytes,35 presumably because of the concentration of RyR at the t-tubules, and occur uniformly across the cell width.53 These data suggest that such release occurs uniformly throughout the transverse, but not longitudinal, elements of the t-tubule network, compatible with the localization of RyRs. However, in detubulated myocytes Ca2+ sparks occur predominantly at the cell surface, and decrease at distances >2 μm from the cell edge.53 Thus, disruption of the t-tubules appears to inhibit Ca2+ sparks within the cell, despite unaltered SR structure and uniform SR Ca2+ load throughout the cell.

Spark frequency is not altered in control or detubulated cells by inhibition of trans-sarcolemmal Ca2+ flux.53 The question therefore arises: why do sparks occur predominantly at membranes bordering the extracellular space? One possible explanation is that the ultrastructure of the dyad is important. Dyadic structure at the surface membrane appears to be unaffected by detubulation; however following detubulation, the t-tubules reorient more longitudinally,17 so that t-tubule dyads are likely to be disrupted. In atrial myocytes, RyRs in the cell centre, which are not associated with Ca2+ channels, have a lower spark frequency than those at the cell surface, but their spark frequency can be increased by a peptide segment of the LTCC,57 and during development Ca2+ sparks appear in rat ventricular myocytes when LTCCs and RyRs co-localize.58 It seems possible therefore that the decrease in RyR opening, and hence spark frequency, in the centre of detubulated myocytes is due to dyad disruption. Thus, spontaneous SR Ca2+ release, which is more marked in conditions of Ca2+ overload, appears to occur where Ca2+ can be rapidly extruded from the cell, helping to keep the cell in Ca2+ balance. Non-junctional RyRs appear to have a lower propensity for initiating such events, although they can contribute to propagating Ca2+ waves.59 It is possible that Ca2+ depletion within the t-tubules, for example at high stimulation rates, might reduce Ca2+ entry and thus help decrease the risk of Ca2+ overload, spontaneous Ca2+ release, and hence arrhythmogenic delayed afterdepolarizations due to the resultant activation of NCX.

The effect of PKA-dependent RyR phosphorylation on RyR opening probability in vivo is controversial. It does not appear to stimulate Ca2+ sparks in intact myocytes,60 although it may alter the kinetics of ICa-triggered Ca2+ release.61 This is consistent with bilayer recordings showing that PKA enhances RyR sensitivity to rapid changes in [Ca2+], but not to steady-state resting [Ca2+].62 Under some conditions, particularly of Ca2+ overload, Ca2+ sparks can initiate a wave of propagated Ca2+ release within the cell, which is dependent on SR Ca2+ load and RyR organization, and may also be enhanced by RyR phosphorylation and intact dyads. Thus, Ca2+ wave propagation is slower in detubulated myocytes, possibly as a result of dyad disruption.

3. Ca2+ uptake by sarcoplasmic reticulum

3.1 Loading via ICa

In addition to triggering Ca2+ release from the SR, ICa loads the SR with Ca2+ for subsequent release.43 Measurements of peak ICa show ∼75% of ICa in the t-tubule membrane;28 however because of slower inactivation of ICa in the surface membrane,34 Ca2+ entry across the surface membrane is greater than might be expected from measurements of peak ICa (e.g. during an action potential in a rat ventricular myocyte, 1.4 µM enters across the surface membrane vs. 3.0 µM across the t-tubule membrane).28 Normalizing to junctional area shows that Ca2+ entry/µm2 of junction is actually higher in the surface membrane than at the t-tubules as a result of the slower inactivation (e.g. 1.4 nM in the surface membrane vs. 1.1 nM in the t-tubule membrane during a rat action potential). Importantly, the extra Ca2+ entry across the surface membrane occurs during the latter part of ICa, which has been proposed to be important in loading the SR with Ca2+,43 suggesting that SR Ca2+ loading via ICa may occur predominantly at the surface membrane.

This idea is supported by experiments investigating the effect of detubulation on SR Ca2+ loading. Following application of caffeine to deplete the SR, the rate of recovery of SR Ca2+ load, and hence Ca2+ transient amplitude, is slower in detubulated cells than in intact myocytes.53 However, SR reloading is slowed by a factor of <2, which more closely reflects the loss of Ca2+ entry than the decrease in amplitude of ICa. Interestingly, in detubulated cells during recovery, the Ca2+ transient occurred initially only at the cell periphery, but during subsequent stimuli, the velocity and extent of Ca2+ propagation into the cell increased.53 This suggests that propagation via Ca-induced Ca2+ release increases as SR Ca2+ content increases. Caffeine-induced Ca2+ release, and hence SR Ca2+ content, appeared to be the same across the width of the cell suggesting that the increasing propagation was not due to spatial differences in SR Ca2+ content.53 It also suggests that Ca2+ entering the cell at the surface membrane does not cause preferential loading of the peripheral SR, but rather that SR Ca2+ content increases throughout the cell, consistent with suggestions that intra-SR Ca2+ equilibrates rapidly between regions of the SR.

Ca2+ entry following detubulation is reduced by 60%,28 a value close to the fraction of NCX present in the t-tubules (63%).49 Since Ca2+ entry via ICa is extruded by NCX during a normal Ca2+ cycle,5 this can explain how SR Ca2+ load remains constant after detubulation, and may help reduce changes in [Ca2+] in the t-tubule lumen. However, modelling suggests that at high stimulation rates, Ca2+ extrusion is slightly less than influx across the t-tubule membrane so that at steady-state lumenal Ca2+ concentration is below bulk cytoplasmic [Ca2+].50 This may decrease ICa, but also implies that under these conditions there must be a net uni-directional Ca2+ flux through the cell, from the t-tubule to the surface membrane.

3.2 Effect of sarcoplasmic reticulum Ca2+ ATPase and Na+/Ca2+ exchange activity on Ca2+ loading

The Ca2+ load of the SR will depend not only on ICa, but also on the relative activity of SERCA and NCX, which compete to remove Ca2+ from the cytoplasm.1 An increase in SERCA activity, or a decrease in NCX activity, will increase SR Ca2+ content, and hence Ca2+ transient amplitude.

SERCA is regulated by PLB, which in its dephosphorylated state inhibits SERCA. Phosphorylation of PLB by PKA (at Ser16) or CaMKII (at Thr17) results in detachment of PLB from SERCA, accelerating Ca2+ uptake.12 Although biochemical data suggest that SERCA and PLB are distributed throughout the SR membrane,63 immunohistological studies show SERCA2b and PLB located predominantly at the Z-line, adjacent to the t-tubules.64 However, detubulation has no effect on the phosphorylation of Ser16 or Thr17 of PLB in response to the beta-adrenergic agonist isoprenaline.52 This suggests that PLB is being phosphorylated by a ‘global’ cell signal, rather than localized signalling like ICa.

NCX is also located predominantly in the t-tubule membrane.41,49 It may play a role in Ca2+ influx, particularly during the early phase of the action potential.65 However, its major role appears to be as a Ca2+ efflux pathway. Its location at the t-tubules may help ensure rapid and synchronous Ca2+ removal from the cytoplasm throughout the cell and may help limit Ca2+ depletion in the t-tubules as a result of ICa in the t-tubule membrane. As the major Ca2+ extrusion pathway, NCX plays an important role in determining cellular, and hence SR Ca2+ content. The regulation of NCX by phosphorylation is controversial.15 However, changes in membrane potential and in trans-sarcolemmal Na+ and Ca2+ gradients will alter its activity, so that a rise in intracellular Ca2+ will increase Ca2+ efflux, whereas an increase in intracellular Na+ or depolarization of the cell membrane will decrease Ca2+ efflux.66

NCX may have privileged access to Ca2+ released from the SR: measurement of INCX and bulk cytoplasmic [Ca2+] during SR Ca2+ release has shown that INCX develops and decays more quickly than the accompanying changes in bulk cytoplasmic [Ca2+].48 This suggests that NCX may exist in a compartment that has restricted diffusion with the bulk cytoplasm, but is closely associated with Ca2+ release and uptake by the SR. If so, NCX is situated to extrude Ca2+ released from the SR, and to compete with Ca2+ sequestration in the SR by SERCA. This may reflect the location of the majority of NCX in the t-tubule membrane, adjacent to, but not at, the dyad.24 The t-tubules may also influence NCX activity because the concentration of (particularly) inward currents in the t-tubule membrane means that they play an important role in determining action potential configuration and hence NCX function.

Thus, NCX may access a Ca2+ pool that has restricted diffusion with the bulk cytoplasmic compartment and is shared by RyR and SERCA. Since these proteins appear to be located predominantly at the t-tubules, such Ca2+ pools may occur predominantly at the t-tubules. PLB may be predominantly similarly located, where it can regulate SERCA close to the t-tubule membrane, and hence RyR. Although immunohistological data suggest that the physical co-localization—and hence, presumably, the functional interaction—of these proteins is less marked than that of ICa and RyR, it is tempting to suggest a similar, although less tightly coupled, functional domain, predominantly at the t-tubules, where Ca2+ removal pathways compete for Ca2+ to be removed from the cytoplasm, and thus help determine SR Ca2+ content.

4. t-tubules and altered sarcoplasmic reticulum function in pathological conditions

Myocytes from failing hearts have reduced SR Ca2+ content, and a systolic Ca2+ transient that is smaller and slower than that in control myocytes. Ten years ago, it was speculated that t-tubule remodelling might contribute to the changes in excitation–contraction coupling observed in failing myocytes.67 However, evidence to support this hypothesis is only emerging now, with recent studies of t-tubule structure in living cells from heart failure models and patients. A marked loss of t-tubules in ventricular myocytes has been described in a canine model of tachycardia-induced heart failure68,69 and in a rabbit model of heart failure.70 In failing human myocytes, a prominent t-tubule network has been reported, although this study lacked control data;40 more recently, it has been reported that there is an increase in the size of the t-tubules and in the number of longitudinal extensions, compared with control human heart.71

Relatively subtle changes in the structure of the t-tubules, including the appearance of gaps in the network, have also been reported in mouse and rat models of heart failure (Figure 1).72,73 In these models, the changes were associated with desynchronization of Ca2+ release, due to loss of triggered Ca2+ release in regions of the cell lacking t-tubules: loss of t-tubules may result in altered spatial distribution of LTCCs, while RyR distribution remains normal, resulting in some ‘orphaned’ RyRs.73 These RyRs and/or an increased number of longitudinal t-tubules (Figure 1) are likely to induce a delay in SR Ca2+ release, as ICa-triggered Ca2+ release is propagated to orphaned RyRs, resulting in desynchronized SR Ca2+ release,72,73 and thus reduced amplitude and slower time course of the systolic Ca2+ transient, and hence contraction.40 The same subtle t-tubule reorganization has been observed in failing human heart71 and computer modelling has shown that this can reduce the synchrony of Ca2+ spark production and lead to late Ca2+ sparks and greater non-uniformity of intracellular Ca2+,71 as observed in animal models of heart failure.74,75 These data suggest that geometric factors can play an important role in the pathophysiology of human heart failure. This is consistent with the decreased SR Ca2+ release for a given ICa observed in a rat model of heart failure which, it was speculated, was due to t-tubule remodelling.67,76 In addition to desynchronization of triggered Ca2+ release, an increase in the amplitude of Ca2+ sparks has been reported during cardiac hypertrophy,77 also attributed to alterations in the dyad. Such an increase in spark amplitude may increase the probability of regenerative Ca2+ waves occurring, although their ability to generate arrhythmias will depend on access to NCX, and thus on t-tubule structure and ultrastructure. Changes in t-tubule structure, and hence the proximity of any given region of cytoplasm to Ca2+ extrusion pathways in the t-tubule membrane, might also be expected to slow Ca2+ extrusion via NCX and alter the balance between SR and sarcolemmal Ca2+ removal. Thus, changes in t-tubule structure in heart failure may alter triggered and spontaneous SR Ca2+ release and SR Ca2+ uptake, although it remains to be explored whether this occurs in other cardiac disease states.

Figure 1

Changes in t-tubule morphology in failing rat ventricular myocytes. (A) Confocal image of a (control) myocyte from a Wistar-Kyoto (WKY) rat, stained using Di-8-ANEPPS. (B) Myocyte from an age-matched spontaneously hypertensive/heart failure rat (SHR) imaged as in A. (C and D) Magnified views of A and B, respectively, with high-contrast look-up table. (E and F) t-tubule line tracings from C and D, respectively. (G) Power vs. spatial frequency along the longitudinal axis computed using Fourier analysis. Failing myocytes display a decrease in power at 0.5 µ m−1, which corresponds to the average t-tubule spacing of 2 µm seen in control cells. The 2nd and 3rd harmonic components seen in WKY are almost completely absent in the SHR cells, consistent with the chaotic appearance of t-tubules in these cells. (H and I) Density of longitudinal elements (LEs; H) and transverse elements (TEs; I). Bars represent the percentage of cell pixels positive for Di-8-ANEPPS staining that were part of a continuous line of stained pixels extending for at least 2 µ m in either the longitudinal (H) or the transverse (I) direction. ***P < 0.001 vs WKY controls, n = 9–13 cells from 4 hearts. Taken from,73 Copyright 2006 National Academy of Sciences, USA.

In addition to changes of t-tubule structure, the distribution of ion channels and transporters between the surface and t-tubule membranes may change: changes in the functional distribution of LTCC and NCX may alter the efficacy of SR Ca2+ release and Ca2+ removal. It has been shown that there are changes of LTCC expression, and isoform switching of the α1C subunit of LTCC, in failing heart,78 although their distribution remains unknown. In addition, expression and current density of NCX is increased in most hypertrophy and heart failure models.79 This supports the hypothesis that increased expression of NCX in the failing human myocardium promotes enhanced extrusion of Ca2+ from the myocyte, and that this (with reduced expression of SERCA) contributes to the reduction in SR Ca2+ content observed in the failing myocardium.9 It should be noted, however, that up-regulation of NCX in heart failure can improve contractile function80 but may also lead to arrhythmias.81 Whether NCX keeps its preferential localization at the t-tubules remains to be determined. It seems likely, however, that changes in t-tubule structure and protein (co-) localization contribute to the changes in SR function observed during heart failure.

Funding

The authors are grateful to the British Heart Foundation and Wellcome Trust for funding.

Acknowledgements

FB is a Wellcome Trust Career Development Fellow.

Conflict of interest: none declared.

References

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