Download T-Tubule Function in Mammalian Cardiac

Review
T-Tubule Function in Mammalian Cardiac Myocytes
Fabien Brette, Clive Orchard
Abstract—The transverse tubules (t-tubules) of mammalian cardiac ventricular myocytes are invaginations of the surface
membrane. Recent studies have suggested that the structure and function of the t-tubules are more complex than
previously believed; in particular, many of the proteins involved in cellular Ca2⫹ cycling appear to be concentrated at
the t-tubule. Thus, the t-tubules are an important determinant of cardiac cell function, especially as the main site of
excitation-contraction coupling, ensuring spatially and temporally synchronous Ca2⫹ release throughout the cell.
Changes in t-tubule structure and protein expression occur during development and in heart failure, so that changes in
the t-tubules may contribute to the functional changes observed in these conditions. The purpose of this review is to
provide an overview of recent studies of t-tubule structure and function in cardiac myocytes. (Circ Res. 2003;92:11821192.)
Key Words: cardiac muscle 䡲 t-tubules 䡲 excitation-contraction coupling 䡲 heart failure
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T
longitudinal extensions.13 Although the t-tubules leave the
surface membrane at the Z line, forming an approximately
rectangular array, only ⬇60% of the tubular volume occurs
near the Z line; the other 40% occurs between the Z lines.2
Thus, the t-tubular system is not, as its name might suggest,
a simple transverse system of tubules but is a complex system
of branching tubules with both transverse and longitudinal
elements (Figure 1). Because of its complexity, it has been
suggested that the transverse-axial tubular system,14 sarcolemmal Z rete,2 or sarcolemmal tubule network11 might be
a more appropriate name. However, t-tubule remains the
standard term and will be used in the present review, and
surface sarcolemma will be used to describe that part of the
cell membrane not within the t-tubules.
Estimates of the percentage of the ventricular myocyte
volume occupied by the t-tubules varies from 3.6% in rat
myocytes2 to 0.8% in mouse myocytes,7 although this variation probably reflects methodological rather than species
differences because there appears to be no clear relationship
between species and estimates of percentage cell volume
occupied by the t-tubules. Similarly, estimates of the percentage of cell membrane located in the t-tubules vary from 64%
(calculated in Bers11 from data in Soeller and Cannell2) to
21%,15 with no apparent species differences.
The t-tubules of cardiac muscle have a mean diameter of
⬇200 to 300 nm,13 although within a single rat ventricular
myocyte, the diameter of individual tubules can vary from 20
to 450 nm, but with more than half the t-tubules having
diameters between 180 and 280 nm.2 One consequence of this
network of narrow tubules is that a rapid change in the
composition of the solution around a ventricular myocyte
results in a slower change in the composition of the fluid
within the t-tubules because of the time required for diffusion
he transverse tubules (t-tubules) of mammalian cardiac
ventricular myocytes are invaginations of the surface
membrane that occur at the Z line and have both transverse
and longitudinal elements. Many of the proteins involved in
excitation-contraction coupling appear to be concentrated at
the t-tubules. Therefore, it has been suggested that the
t-tubules play a central role in cell activation. In the present
review, we will consider the immunohistochemical and functional evidence for protein localization at the t-tubules,
potential problems in the interpretation of such data, and the
functional consequences of such localization. We will also
consider the possible role of the t-tubules in the functional
changes that occur during cardiac development, hypertrophy,
and failure.
Occurrence and Morphology of the T-Tubules
T-tubules are present in the cardiac tissue of all species of
mammals so far investigated (eg, mice,1 rats,2 guinea pigs,3
rabbits,4 dogs,5 pigs,1 and humans6) but appear to be absent in
avian,7 reptile8 and amphibian8 cardiac tissue. Within mammalian cardiac tissue, t-tubules occur predominantly in ventricular myocytes, being either absent or far less developed in
atrial, pacemaking, and conducting tissue,9 although a recent
report has suggested that ⬇50% of atrial myocytes possess a
sparse irregular tubular system.10 The following discussion
will concentrate on mammalian ventricular myocytes.
The t-tubules are invaginations of the sarcolemma and
glycocalyx, which appears to remain associated with the
sarcolemma within the t-tubules.11 Early studies of cardiac
muscle showed that they occur at the Z line, at the end of each
sarcomere12; therefore, they occur at intervals of ⬇2 ␮m
along the longitudinal axis of the ventricular myocyte. Subsequent studies have shown that the t-tubular system also has
Original received March 3, 2003; revision received April 22, 2003; accepted April 22, 2003.
From the School of Biomedical Sciences, University of Leeds, Leeds, UK.
Correspondence to Clive Orchard, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000074908.17214.FD
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Brette and Orchard
Mammalian T-Tubule Function
1183
despite the forces exerted during the normal contraction cycle
may be due to the presence of a “scaffold” of focal adhesion
molecules, membrane-associated proteins, and basal lamina
proteins.20
Proteins Present in the T-Tubule Membrane
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Figure 1. Three-dimensional structure of the t-tubular system in
a living rat ventricular myocyte, reconstructed from a stack of
images of dextran-linked fluorescein fluorescence within the
t-tubules. Bar⫽5 ␮m. Reprinted from Soeller C, Cannell MB.
Examination of the transverse tubular system in living cardiac
rat myocytes by 2-photon microscopy and digital image–processing techniques. Circ Res. 1999;84:266 –275, by permission
of the American Heart Association ©1999.
into the t-tubular network. In guinea pig myocytes, a rapid
change of [Ca2⫹]o results in the diffusion of Ca2⫹ into the
t-tubules at 3 to 16 ␮m/s,16 so that the solution change within
the t-tubules is delayed by up to 2.3 seconds, and washout of
Ca2⫹ from the t-tubules occurs with a time constant of up to
1.7 seconds for the deeper regions of the t-tubular system,16
although these time courses may vary between species,17
possibly reflecting species differences in t-tubule
morphology.
Although the diversity of estimates of the extent of the
t-tubules within a species makes it impossible to determine
whether there may be differences between species, it is
tempting to speculate that the extent of the t-tubules varies
between species depending on cell size (surface area/volume
ratio) and heart rate if the t-tubules are necessary to produce
synchronous Ca2⫹ release throughout the cell (see Coupling
of Ca2⫹ Entry and Ca2⫹ Release, later) and, hence, synchronous contraction. Although we are unaware of any differences related to cell size, the density of the t-tubular network
is greater in the mouse, which has a resting heart rate of 300
to 400 bpm, than in the pig, which has a heart rate of ⬍100
bpm.1
T-Tubule Development Is Labile
Ventricular myocytes isolated from neonatal hearts show
little evidence of t-tubule development,4,18 and cells kept in
culture for 6 days show a progressive decrease of t-tubule
density.19 T-tubule structure has also been reported to change
in myocytes from failing hearts (see T-Tubule Development
and Morphology in Hypertrophy and Failure). However, the
mechanisms underlying the expression and maintenance of
the t-tubules are not clear. Once formed, the ability of the
tubular system to maintain its remarkable degree of structure
The function of the t-tubules depends not only on their
structure but also on the proteins within, and adjacent to, the
t-tubule membrane. Immunohistochemical techniques have
been widely used to investigate the location of proteins within
cardiac ventricular myocytes. Such studies have shown
marked variations in the distribution of membrane proteins,
although a distinction needs to be drawn between the fraction
of a particular type of protein located at the t-tubule and
whether this is greater than the fraction of the total cell
membrane located within the t-tubules, ie, whether protein
density is higher (concentrated) at the t-tubule. This is
discussed further in Interpretation of Immunohistochemical
Data and in Conclusions and Unanswered Questions.
Ca2ⴙ-Handling Proteins
The location of sarcolemmal Ca2⫹-handling proteins is important because of their role in excitation-contraction coupling and because the Ca2⫹-release channels (ryanodine
receptors [RyRs]) of the sarcoplasmic reticulum (SR) are
concentrated close to the t-tubule (see Protein Colocalization
Within, and With Proteins Adjacent to, the T-Tubules).
In the rabbit heart, an early study showed that a membrane
fraction from the t-tubules had a higher density of L-type
Ca2⫹ channels than did membrane from the surface sarcolemma.21 A more recent immunologic study also found this
channel to be concentrated in the t-tubules of adult rabbit
ventricular myocytes, with less staining of the surface sarcolemma, although the surface staining that did occur was
punctate and associated with junctional SR.22 In rat heart, the
L-type Ca2⫹ channel, and hence current (ICa), is also concentrated in the t-tubules, with estimates ranging from 3 to 9
times more concentrated in the t-tubule membrane than on the
surface sarcolemma.23–26 Comparative studies suggest that
the t-tubular concentration of the L-type Ca2⫹ channel is
greater in rat ventricular myocytes than in those from the
rabbit.22
Although a high density of L-type Ca2⫹ channel at the Z
line has been reported in all studies of which we are aware, it
has also been reported that in some rat ventricular myocytes,
the channel is present in appreciable amounts on the surface
membrane,25 and in sheep and bovine cardiac tissue, it has
been reported that this channel is present at a lower concentration on the putative t-tubule membrane than on the surface
sarcolemma.27 However, the majority of recent reports suggest that this channel is concentrated in the t-tubules compared with the surface sarcolemma.
The distribution of the Na⫹-Ca2⫹ exchanger (NCX) has
been more controversial. The first study of its distribution
showed it to be predominantly in the t-tubules in guinea pig
ventricular myocytes.28 Subsequent studies (eg, Musa et al25
and Kieval et al29) have shown a more even distribution
between the t-tubule and surface membranes, although a
recent immunologic study of rat ventricular myocytes also
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Figure 2. Schematic diagram of protein distribution between the t-tubules and surface sarcolemma. See text for further explanation.
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showed NCX located predominantly within the t-tubular
network.26 Thus, although there is a consensus that the NCX
protein is found in the t-tubule membrane, it is less clear
whether this protein is at a higher concentration in the
t-tubules or is homogeneously distributed between the
t-tubules and surface sarcolemma.
The distribution of other important sarcolemmal Ca2⫹handling proteins, in particular, the sarcolemmal Ca2⫹ATPase and the T-type Ca2⫹ channel, is currently unknown.
Naⴙ-Handling Proteins
In addition to NCX, other proteins that allow Na⫹ to cross the
cell membrane also show different distributions between the
t-tubules and surface sarcolemma. For example, the Na⫹-H⫹
exchanger, which regulates intracellular pH by extruding H⫹
from the cell, is concentrated at the t-tubules and intercalated
disks.30 Immunolabeling of the Na⫹,K⫹-ATPase has shown
that different isoforms are differently distributed, with the ␣1
isoform being concentrated in the t-tubules of rat ventricular
myocytes and the ␣2 isoform showing a more homogeneous
distribution.31 In contrast, in guinea pig myocytes, which only
express the ␣1 isoform, this isoform showed a more uniform
distribution between the t-tubule and surface sarcolemma.31
The Na⫹ channel has been found to be localized to the
t-tubules in ventricular myocytes (see Scriven et al26). A
recent immunologic study has shown that the cardiac ventricular myocyte expresses several different Na⫹ channel isoforms, with the “cardiac” pore-forming subunit Nav1.5 located predominantly at the intercalated disks but with the
“brain” isoforms Nav1.1, Nav1.3, and Nav1.6 expressed predominantly in the t-tubules.32 However, the function of these
isoforms is unclear, because their high tetrodotoxin sensitivity (nanomolar) is in contrast with the high concentrations of
tetrodotoxin (micromolar) required to affect cardiac
preparations.11
Thus, the t-tubules may represent a region of the cell where
the regulation of Na⫹ is different from that of the surface
sarcolemma, although the differences appear less marked
than for Ca2⫹-handling proteins. Changes in [Na⫹]i, by
altering the activity of NCX, may have marked effects on
[Ca2⫹]i and, hence, contractility, although this may also
depend on the proximity of the Na⫹-handling proteins to
NCX within the t-tubule (see Protein Colocalization Within,
and With Proteins Adjacent to, the T-Tubules).
Kⴙ-Handling Proteins
There are relatively few studies of the distribution of proteins
carrying K⫹ ions. Barry et al33 observed Kv4.2, Kv1.2, Kv1.5, and
Kv2.1 concentrated at the surface membrane of rat ventricular
myocytes, with labeling intensity highest at the intercalated
disk. However, as pointed out in Takeuchi et al,34 the
resolution of staining in that study was relatively low;
subsequently, Kv4.2, one of the channels that underlies the
transient outward current (Ito) in ventricular myocytes, was
shown to be localized predominantly to the t-tubular system.
Similarly, the K⫹ channel TASK-1,35 which may underlie the
steady-state outward current (Iss), and Kir2.1,36 which is believed to underlie the inward rectifier K⫹ current (IK1), also
appear to be predominantly in the t-tubules.
Anion-Handling Proteins
In addition to Na⫹-H⫹ exchange, another pH-regulating
protein, the Cl⫺-HCO3⫺ exchanger, is present, and may be
concentrated, at the t-tubule.37 It is tempting to speculate that
it is important that the pH of this region of the cell, which
contains so many proteins important for normal cell function,
is tightly regulated.30
Second-Messenger Pathways
The ␤-adrenergic pathway is important in the regulation of a
number of key proteins in the heart, in particular, the L-type
Ca2⫹ channel, the SR Ca2⫹-ATPase (via the regulatory protein
phospholamban), and the contractile proteins (via troponin I).
It has been suggested that there is local regulation by this
pathway, particularly of the L-type Ca2⫹ channel.38 A structural basis for such regulation is suggested by reports that key
elements in this signaling cascade (Gs, adenylate cyclase, and
A-kinase anchoring protein) are concentrated at the t-tubule
membrane.39 – 41 Interestingly, the protein phosphatase calcineurin, which will antagonize the effects of this pathway,
appears to be colocalized with protein kinase A (PKA) near
the t-tubules.42 The ␤2-receptor may also be associated with
the L-type Ca2⫹ channel, because in neurons this receptor has
been shown to be part of a macromolecular signaling complex with the L-type Ca2⫹ channel Cav1.2, a G protein, and
Brette and Orchard
adenylate cyclase.43 This could account for the observation
that stimulation of this receptor can increase ICa without an
increase in whole-cell cAMP concentration (see Signal
Transduction). Such a localized response has also been shown
for the NO synthase 3 pathway.44
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Interpretation of Immunohistochemical Data
Data from these studies show many key proteins concentrated
at the t-tubule (Figure 2). However, there are a number of
potential problems with immunohistochemical studies. First,
the observed distributions may be artifactual. It has, for
example, been suggested that high-intensity staining at the
intercalated disks and t-tubules may be a consequence of
membrane folding and a high “brightness factor,” respectively, so that quantification of protein distribution from such
studies is difficult.45 Second, the observed distribution may
depend on epitope accessibility rather than protein distribution,46 and spatial resolution is limited; further immunogold
labeling may yield more information. Third, it is not always
clear that a “striated” pattern of cell staining is colocalized
with the t-tubule membrane or that the protein is inserted in
the membrane. Therefore, it is difficult to be sure that
particular proteins are concentrated at the t-tubules, particularly when the amount of membrane within the t-tubules is
also unclear.
It is interesting, however, that so many proteins are
identified at the Z line, which, if correct, raises questions of
whether these proteins function at the t-tubules or whether
they are simply trafficked via this region of the cell. It is not
clear that protein location always corresponds to function,
partly because of some of the problems mentioned above and
partly because protein function will also depend on other
factors, such as local environment, second messengers, and
accessory proteins. Localization of protein function will be
considered further (in Localization of Function). Concentration of many proteins at the t-tubules may reflect their
importance in excitation-contraction coupling (see Coupling
of Ca2⫹ Entry and Ca2⫹ Release), although this will depend on
not just the presence of the proteins but their juxtaposition
(see Protein Colocalization Within, and With Proteins Adjacent to, the T-Tubules, below).
Protein Colocalization Within, and With Proteins
Adjacent to, the T-Tubules
The SR is the major intracellular Ca2⫹ store in cardiac muscle.
Ca2⫹ entry across the cell membrane triggers the release of
further Ca2⫹ from the SR, via the RyR,47 and it is predominantly this Ca2⫹ that causes contraction.
RyRs were first reported as “feet”48 between the sarcolemma and SR membrane. Immunologic studies have shown
a high density of RyRs in the junctional SR (the Ca2⫹-release
site) adjacent to the t-tubule.23,26 Thus, the site of Ca2⫹ release
from the SR appears to be concentrated at the t-tubule,
adjacent to the site of transsarcolemmal Ca2⫹ flux (see
Ca2⫹-Handling Proteins).
Interestingly, the SR Ca2⫹ uptake pump (SERCA2), which
is responsible for removing Ca2⫹ from the cell cytoplasm to
cause relaxation and which, according to structural and
biochemical studies (eg, Jorgensen et al49), is thought to be
Mammalian T-Tubule Function
1185
located throughout the SR membrane, has been shown
immunohistochemically to be concentrated at the Z line,
adjacent to the t-tubule.25 The regulatory protein phospholamban, which modulates the activity of SERCA2, shows a
similar distribution (F. Brette, unpublished data, 2002).
Immunologic investigation of colocalization of proteins
involved in excitation-contraction coupling has shown that
Ca2⫹ channels are highly colocalized with RyRs in the
t-tubules, forming the dyad.23,26 However, NCX shows little
colocalization with either the RyR or Na⫹ channel.26 Therefore, it has been suggested that the t-tubule can be subdivided
into 3 domains26: (1) the dyad, (2) one containing the Na⫹
channel, and (3) one containing NCX. This suggests that the
most effective functional coupling will be between the L-type
Ca2⫹channel and the RyR, although the mechanism of colocalization is still unknown. The lack of colocalization of NCX
with RyR may be one reason why Ca2⫹ influx via NCX is a
less effective trigger for SR Ca2⫹ release.50,51 The lack of
colocalization of NCX and Na⫹ channels is also interesting,
because it has been suggested that Na⫹ influx via Na⫹
channels may enhance Ca2⫹ entry on the exchanger,52 a
mechanism that would presumably require colocalization;
however, the proximity of NCX to junctional SR may allow
NCX activity to be modulated by Ca2⫹ released from the SR
(see Trafford et al53 and Ca2⫹ Efflux).
Localization of Function
The studies described above suggest that many membrane
proteins are concentrated at the t-tubules. However, protein
function will not necessarily reflect protein distribution
(above). A different approach has been to investigate the
localization of function by using a number of techniques,
which follow.
Cells Lacking T-Tubules
Some cardiac cells do not have a t-tubular system or have
only a very sparse system; these include ventricular myocytes
from embryonic and newborn animals, atrial cells, and
Purkinje cells.4,9,54,55 In addition, adult ventricular myocytes
kept in culture lose their t-tubular system.19,56 A number of
studies have investigated the function of these cell types,
which may help our understanding of t-tubule function.
In myocytes lacking t-tubules (from the ventricles of
newborn rabbits, atrial cells, rabbit Purkinje cells, and cultured ventricular myocytes), electrical stimulation causes a
rise of [Ca2⫹]i that occurs initially at the cell periphery and
propagates into the cell interior by propagated Ca2⫹-induced
Ca2⫹ release in atrial55 and cultured56 cells and by diffusion in
neonatal4 and Purkinje54 cells, and localized Ca2⫹-release
events (Ca2⫹ sparks) occur predominantly close to the surface
sarcolemma in these cells.56 This is in contrast to adult
ventricular myocytes, in which Ca2⫹ release occurs synchronously across the width of the cell on electrical stimulation,4,57,58 and Ca2⫹ sparks arise predominantly at the
t-tubule.59 These data are consistent with the notion that
t-tubules play an important role in causing synchronous Ca2⫹
release in the adult ventricular myocyte, because of the
concentration of L-type Ca2⫹ channels and RyR at the t-tubule
(see Ca2⫹-Handling Proteins, above), although a problem
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with this type of study for the investigation of t-tubule
function is that protein expression, as well as t-tubule structure, may be different in these cells.
An alternative approach has been to correlate the loss of
t-tubules with loss of membrane currents. During 4 days in
culture, the membrane capacitance of rabbit ventricular myocytes, taken to represent membrane area, decreases by 51%
with a time course that correlates with the loss of 83% of IK1
and 88% of the ATP-sensitive K⫹ current (IK,ATP),60 suggesting that these currents are concentrated in the t-tubules.
However, another study has shown more complicated
changes in rabbit ventricular myocytes during culture19:
although t-tubule density and cell capacitance decrease continuously during 6 days in culture, Ca2⫹ channel density
decreases by ⬇50% after 1 day and then partially recovers,
IK1 also declines by ⬇50% within 1 day but shows no
recovery, whereas Ito changes little in 1 day but decreases by
65% after 6 days, emphasizing the difficulty of distinguishing
between changes due to loss of the t-tubules and changes in
protein expression.
Diffusion Studies
A different approach has been to investigate the rate of
change of membrane currents in ventricular myocytes after a
rapid change in perfusate composition. Because of the long
diffusion time into the t-tubules (see Occurrence and Morphology of the T-Tubules), currents on the cell surface would
be expected to change rapidly, whereas those located in the
t-tubules would be expected to change over a longer time
course. By use of this technique, it has been shown that the
time course of K⫹ current inhibition by Ba2⫹ is consistent
with a model that includes a diffusion time constant of 300
ms, consistent with much of IK1 and IK,ATP being localized in
the t-tubules.60 A similar approach has been used in guinea
pig myocytes to investigate the time course of changes of the
Na⫹ current (INa) and ICa in response to rapid changes of
extracellular Na⫹ and Ca2⫹, respectively.61 In atrial cells,
which lack t-tubules, changing the bathing Na⫹ or Ca2⫹
produced rapid changes of INa and ICa (time constant of ⬇25
ms). However, in ventricular myocytes, only 36% of the
current changed rapidly; the remaining 64% of current
changed with a time constant of ⬇200 ms, suggesting that
this percentage of INa and ICa is within the t-tubules. Interestingly, this percentage is the same as the upper estimate of the
percentage of cell membrane within the t-tubules (see Occurrence and Morphology of the T-Tubules), which would imply
that these currents are found predominantly, but not concentrated, within the t-tubules.
Scanning Pipette
An elegant technique has been described recently that uses a
pipette to scan the cell surface and simultaneously monitor
membrane currents.62 To date, this technique has been used to
investigate the location of IK,ATP, which appears to be concentrated in the vicinity of the Z line, consistent with the proposal
(see Diffusion Studies) that this current is found predominantly in the t-tubule. However, it is not clear whether the
current monitored at the Z line by this technique simply
reflects the amount of membrane under the electrode (ie, in
the t-tubule) or whether the current is concentrated in the
t-tubule or concentrated on the surface sarcolemma at the Z
line.
Detubulation
Another approach developed recently has been to adapt the
“osmotic shock” technique used previously to detubulate
skeletal muscle to disrupt the t-tubules of rat ventricular
myocytes.24 Detubulation decreases cell capacitance by
⬇30%.24 It is not clear that the standard method used to
measure membrane capacitance (eg, 10-mV 10-ms hyperpolarizing pulses from ⫺80 mV) monitors the capacitance of all
the t-tubule membrane (see Electrical Properties of
T-Tubules). However, assuming the capacitance of the
t-tubule membrane to be the same as that of the surface
sarcolemma, this gives a lower limit to the percentage of the
cell membrane found within the t-tubules.
Most (⬇87%) of ICa and almost all NCX activity are lost
after detubulation,24,58 suggesting that the function of both of
these Ca2⫹ flux pathways is concentrated in the t-tubules of
rat ventricular myocytes, although if cell capacitance is
underestimated more than ionic currents, it is possible that
these currents are concentrated less than these measurements
might suggest (see Electrical Properties of T-Tubules). In
contrast, INa shows uniform distribution between the t-tubule
and surface membranes,58 as do IK, Ito, and IK1, although Iss
appears to be concentrated in the t-tubules.63 This distribution
of Iss agrees with immunohistochemical studies of TASK-1
(see K⫹-Handling Proteins). However, the proteins thought to
underlie Ito and IK1 appear to be concentrated in the t-tubules
(see K⫹-Handling Proteins); this discrepancy may be due to
the difficulties of these techniques (see Interpretation of
Immunohistochemical Data, above, and Electrical Properties
of T-Tubules, below) or to local regulation or differential
distribution of accessory proteins such as Kv4.3,63 which, with
Kv4.2, forms functional Ito channels.
Detubulation shows a greater percentage of Ca2⫹ flux via
ICa and NCX in the t-tubules than might be expected from
immunohistochemical studies. One way of reconciling these
data is to suggest that the protein in the t-tubule is more active
than that in the surface membrane (because of stimulation of
ICa by second-messenger pathways, for example), so that
relatively more function is lost than protein after detubulation. In support of this idea, ␤-adrenergic stimulation of ICa
causes a greater increase in current in normal cells than in
detubulated cells,64 suggesting that ICa in the t-tubules might
be better coupled to this second-messenger pathway than that
in the surface membrane. In the presence of tonic activity of
this pathway, this could explain the relatively large loss of
current after detubulation.
Functional Role
Electrical Properties of T-Tubules
Localized depolarization of skeletal muscle at the t-tubule
causes contraction of the adjacent half sarcomeres.65 By
analogy, it has been assumed that the t-tubules in cardiac
myocytes allow propagation of excitation into the cell to
cause synchronous activation, although analogous experiments in cardiac muscle have not shown localized activation,
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Mammalian T-Tubule Function
1187
Figure 3. Visualization of t-tubule–SR
dyadic release. A, Confocal line-scan
image showing discrete t-tubule–SR
Ca2⫹-release events (black arrowheads,
top), mean fluorescence (middle), and ICa
(bottom) on depolarization as shown
above traces. Bar⫽5 ␮m. B, Data from
panel A shown as a 3D plot of fluorescence (F) in time (t) and space (X). Modified from Song LS, Sham JS, Stern MD,
Lakatta EG, Cheng H. Direct measurement of SR release flux by tracking
“Ca2⫹ spikes” in rat cardiac myocytes.
J Physiol. 1998;512:677– 691, by permission of the Physiological Society ©1998.
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possibly because the longitudinal extensions of the t-tubules
allow the spread of excitation to adjacent regions of the cell.66
Tidball et al67 observed that the response of the action
potential of rabbit ventricular trabeculae to the SR inhibitor
ryanodine was correlated with the degree of t-tubule development and postulated that the t-tubules had a density of Ca2⫹
and K⫹ channels that was different from that of the surface
membrane. It now appears likely that there are at least 3
factors that could contribute to differences between the
t-tubule and surface membrane action potentials: (1) many of
the membrane proteins that underlie the action potential are
unevenly distributed between the surface and t-tubule membranes (above), so that the electrophysiology of the t-tubules
would be expected to differ from that of the surface membrane; (2) modeling of skeletal muscle t-tubules has shown
that 2 to 3 ms is required before the potential throughout a
simple t-tubule model matches that at the cell surface,68 and
the complex structure of the cardiac t-tubular system may
result in longer delays; and (3) the t-tubules form a restricted
extracellular space that will allow ion accumulation and
depletion, which can modify electrical activity (see Clark et
al36). In isolated cells, this may differentially alter the t-tubule
action potential; in the intact muscle, diffusion is also
restricted in the intercellular clefts, and the extent to which
diffusion from the t-tubules and the intercellular clefts will
differ is unknown.
Consideration of the electrical properties of the t-tubules is
important (1) because it seems likely that the t-tubules are the
most important site for excitation-contraction coupling and
(2) because standard voltage-clamp techniques may not
effectively clamp potential, and hence monitor membrane
currents, within the t-tubular system.69 Thus, ionic currents at
the surface membrane may be more effectively monitored
than those in the t-tubules, so that loss of currents with loss of
t-tubules (see Cells Lacking T-Tubules and Detubulation)
may be underestimated. Similarly, since the voltage drop
down the t-tubules is unknown, it is possible that measurements of cell capacitance (see Cells Lacking T-Tubules and
Detubulation) underestimate the percentage of cell membrane
within the t-tubules. If capacitance and membrane currents
are underestimated to a similar degree, calculation of the
concentration of these currents in the t-tubules will be the
same. However, if cell capacitance is underestimated more
than ionic currents, these currents may be less concentrated in
the t-tubules than suggested by such measurements.
Coupling of Ca2ⴙ Entry and Ca2ⴙ Release
The data presented above suggest that ICa and NCX activity
occur predominantly in the t-tubules, close to the RyR,
implying that the t-tubules play a central role in Ca2⫹ cycling
and excitation-contraction coupling in cardiac ventricular
myocytes. Although ICa is more effective than NCX in
triggering SR Ca2⫹ release,50,51 probably because the L-type
Ca2⫹ channel, unlike NCX, is colocalized with RyR26 and
because of the higher Ca2⫹ flux through the channel,70 the
presence of NCX in the t-tubules means that Ca2⫹ flux via
NCX may participate in the process.71
The local control theory of Ca2⫹ release (see Wier and
Balke47 for review) is that local Ca2⫹ entry across the cell
membrane, predominantly via ICa, triggers local Ca2⫹ release
from an adjacent cluster of RyRs. The whole-cell Ca2⫹
transient is the temporal and spatial sum of these individual
localized release events.72 Ca2⫹ sparks occur predominantly
near the t-tubules,59,73 and localized Ca2⫹ release (Ca2⫹
spikes) occurs at discrete sites at the Z line.74 These spikes
(Figure 3) are proportional to ICa and to derived SR Ca2⫹ flux,
providing strong support for the idea that local Ca2⫹ entry
across the t-tubule membrane triggers local Ca2⫹ release from
adjacent RyRs.
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 [Ca2⫹]i initially
occurs close to the cell membrane and then either diffuses
(Purkinje54 and neonatal4 cells) or propagates via Ca2⫹induced Ca2⫹ release (atrial,75 cultured,56 and detubulated57,58
cells) into the cell (Figure 4). Interestingly, in detubulated
cells, the speed of propagation is increased by ␤-adrenergic
stimulation76 so that synchronization of Ca2⫹ release is
increased77 even in the absence of t-tubules.76 In addition,
compared with pig myocytes, mouse myocytes show a rapid
and synchronous rise of [Ca2⫹]i.1 This difference appears to
be due to the higher t-tubule density in mouse myocytes.
Therefore, it appears that Ca2⫹ release occurs predominantly
at the t-tubules, which therefore underlie the temporally and
spatially synchronous Ca2⫹ release observed in ventricular
myocytes (see Yang et al58).
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June 13, 2003
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Figure 4. Spatial [Ca2⫹]i gradients in cardiac myocytes. Transverse line scans show that [Ca2⫹]i rises synchronously across the cell
width in a ventricular cell after electrical stimulation (S), whereas in cells lacking t-tubules (detubulated, atrial, and Purkinje), [Ca2⫹]i rises
initially at the cell edge and then propagates into the cell center. Scales have been changed to allow qualitative comparison. Modified
from Cordeiro JM, Spitzer KW, Giles WR, Ershler PE, Cannell MB, Bridge JH. Location of the initiation site of calcium transients and
sparks in rabbit heart Purkinje cells. J Physiol. 2001;531:301–314; Yang Z, Pascarel C, Steele DS, Komukai K, Brette F, Orchard CH.
Na⫹-Ca2⫹ exchange activity is localized in the t-tubules of rat ventricular myocytes. Circ Res. 2002;91:315–322; and Blatter LA, Kockskamper J, Sheehan KA, Zima AV, Huser J, Lipsius SL. Local calcium gradients during excitation-contraction coupling and alternans in
atrial myocytes. J Physiol. 2003;546:19 –31, by permission.
Although NCX is well recognized as an important Ca2⫹
efflux pathway (see Ca2⫹ Efflux), the suggestion that Ca2⫹
entry on NCX at the start of the action potential might trigger
SR Ca2⫹ release78 has been more controversial; most studies
suggest that it is not as effective as ICa (above) and may not be
the normal physiological trigger but rather modulates the
trigger effect of ICa and/or acts as a trigger only under certain
conditions. It has been proposed that Ca2⫹ entry on the
exchanger, and hence its ability to act as a trigger, is enhanced
by colocalization with INa, which will increase the [Na⫹] in a
submembrane microdomain (fuzzy space79) that is sensed by
NCX. However, Scriven et al26 have shown that the L-type
Ca2⫹ channel, NCX, and INa are in different microdomains,
although a demonstration that brain-specific isoforms of the
Na⫹ channel are concentrated in the t-tubules32 may require
the colocalization of these channels with other proteins in the
t-tubule to be investigated.
As well as “feed-forward” from Ca2⫹ entry via L-type Ca2⫹
channels and NCX to the RyR, the proximity of these proteins
suggests that Ca2⫹ released from the SR may “feed back” onto
the sarcolemmal proteins concentrated at the t-tubule (see
Ca2⫹ Efflux, below).
Ca2ⴙ Efflux
There are 2 sarcolemmal Ca2⫹ efflux pathways in ventricular
myocytes: NCX and sarcolemmal Ca2⫹-ATPase. The exchanger appears to be localized within the t-tubules and is
therefore close to the site of SR Ca2⫹ release (above); the
distribution of Ca2⫹-ATPase is unknown, although it is
present on the surface membrane.58 There are at least 2 lines
of evidence to support the idea that Ca2⫹ released from the SR
has “privileged” access to NCX. First, when Ca2⫹ is released
from the SR using caffeine, there is hysteresis between bulk
[Ca2⫹]i and NCX current, with a given current being produced
by a lower bulk [Ca2⫹]i as [Ca2⫹]i is increasing, suggesting
that Ca2⫹ is released close to NCX, resulting in a higher local
[Ca2⫹]i that stimulates NCX.53 Second, Ca2⫹ efflux appears to
occur during systole, compatible with Ca2⫹ release occurring
close to NCX.80 Interestingly, recent immunohistochemical
work has shown that SR Ca2⫹-ATPase also appears to be
localized close to the t-tubule.25
The observation that major Ca2⫹ sequestration pathways
are located at the t-tubule raises the question of why this is so.
The presence of NCX in the t-tubules may allow rapid Ca2⫹
efflux throughout the cell, thus helping to produce synchronous relaxation. However, it also appears that this arrangement will produce futile Ca2⫹ cycling. The reason for this is
unknown, although it has been suggested that by regulating
[Ca2⫹] close to the RyR, NCX may alter the threshold for, and
thus help regulate, SR Ca2⫹ release.71
Signal Transduction
Colocalization of PKA and calcineurin occurs at the t-tubules
(see Second-Messenger Pathways), and a local increase in
cAMP along the Z lines has been shown by using fluorescence
resonance energy transfer (Zaccolo and Pozzan81). These authors
suggested that a localized increase of cAMP occurs near the
t-tubules, although their preparation, embryonic cardiac cells,
lacks t-tubules. However, this suggests that the Z line, rather than
the t-tubules, might provide the scaffold for the cell structure in
this region, and further experiments in adult ventricular myocytes would be of interest.
There is also functional evidence for a local elevation of
cAMP and subsequent protein phosphorylation. Xiao82 has
provided evidence that ␤2-adrenergic stimulation can produce an
increase in ICa without causing phosphorylation of other proteins,
such as phospholamban, compatible with local signaling and
close association of the ␤2 signaling pathway with the L-type
Ca2⫹ channel (see Second-Messenger Pathways). The observation that the L-type Ca2⫹ channel is less sensitive to ␤-adrenergic
stimulation in detubulated cells, although phospholamban phos-
Brette and Orchard
phorylation is unaffected,83 suggests that this local signaling
occurs predominantly in the t-tubules.
Changes During Development and Disease
Data from normal cells suggest that the major difference
between the t-tubule and surface membranes is the concentration of Ca2⫹-handling proteins at the t-tubules. Therefore,
in this section, we will concentrate on these proteins.
T-Tubule Development and Morphology in
Hypertrophy and Failure
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Embryonic and neonatal cardiac myocytes lack t-tubules,4,84
which develop during the first few weeks of life, with the
precise time of development differing between species. In
addition, during cell culture (above) and heart failure (below),
t-tubule morphology changes, indicating that t-tubule structure is labile even in the mature cell.
How the t-tubules develop and are maintained is less clear.
Lee et al85 have shown that expression of amphisin-2, a protein
that can link the plasma membrane and submembranous cytosolic scaffolds in CHO cells, generates narrow tubules that are
continuous with the plasma membrane. The t-tubule membrane
appears to have a distinct protein and lipid composition and is
enriched in cholesterol, which can be used as a tool to separate
t-tubule and surface membranes.86 The development of the
t-tubules appears to depend on protein and lipid and shows
properties that are similar to the development of caveolae, which
requires cholesterol and caveolin-3.87 T-tubules are composed of
interconnected caveolae-like elements, and repeated caveolae
formation in the absence of fission leads to the generation of
t-tubules.88,89 Treatment with amphotericin B, an antibiotic that
binds cholesterol, causes the disruption of t-tubules in C2C12
myotubes.86 This does not exclude the existence of “true”
caveolae (50- to 80-nm-diameter invaginations, see Razani et
al87 for review) along the surface sarcolemma and t-tubule
membrane.11
Despite the evidence that the t-tubules are important in
excitation-contraction coupling, there have been relatively few
studies of this network during pathological conditions. An early
morphological study showed that hypertrophy of rat left ventricle, produced by aortic constriction, results in an increase in the
t-tubule area, which helps maintain the surface area/volume ratio
of the hypertrophied cells.90 However, in rat doxorubicininduced cardiomyopathy, cell capacitance decreased significantly, possibly as a result of t-tubule damage.91 To date, only
one study has examined t-tubule structure in pathological living
cells. He et al5 showed that in canine tachycardia-dilated
cardiomyopathy, t-tubules are lost at each extremity of the cell
but remain intact in the center of the cell (interestingly, a similar
pattern of t-tubule loss has been reported during culture19);
however, cell capacitance increased by 13%, possibly because of
a hypertrophy-induced increase in surface sarcolemma compensating for the loss of t-tubules.
Data from the human heart is equally equivocal. In hypertrophic human heart, the t-tubules appear to be aberrantly
shaped92 or dilated.20 Dilation has been also observed in
failing human heart,93 although the changes were not quantified in either study. In contrast, a preliminary study of
human ventricular myocytes suggests that t-tubule density is
Mammalian T-Tubule Function
1189
not altered in failing hearts,94 whereas another preliminary
report showed decreased t-tubule density in myocytes from
failing human ventricle.6
The diversity of results from animal and human studies may
reflect the diversity of models and conditions studied. Further
investigation of the development, morphology, and composition
of the t-tubules from the failing heart may elucidate both the
regulation of t-tubule structure5,92 and its role in the changes in
function observed in pathological conditions (see Gomez and
colleagues95,96 and Relevance of Changes in T-Tubule Structure
and Protein Expression to Function).
Changes in Protein Expression in Development,
Hypertrophy, and Failure
Ventricular myocytes from newborn animals show little t-tubule
development (see T-Tubule Development and Morphology in
Hypertrophy and Failure) and relatively little SR97 but enhanced
ICa98 and NCX activity99 compared with adult cells. It seems
likely that these cells rely mainly on extracellular Ca2⫹ for
activation.100 The transition to the more mature (SR-dominated)
form of excitation-contraction coupling during the first few
weeks of life is accompanied by SR development, decreased
ICa,98 and t-tubule formation (Haddock et al4 and Chen et al18),
and NCX at least has been reported to appear in the t-tubules as
soon as they are formed.18
Ventricular myocytes from failing hearts show reduced contraction, blunted ␤-adrenergic responsiveness, and myocyte
hypertrophy. For review, see Tomaselli and Marban101; we will
concentrate on changes in the proteins that occur at the t-tubules.
A decrease in the amplitude and the rate of decline of the
systolic Ca2⫹ transient is a consistent finding in failing heart
muscle. This appears to be due to depressed SERCA2
activity, which will slow the rate of decline of the Ca2⫹
transient and decrease SR Ca2⫹ content. In addition, RyRs
may be hyperphosphorylated, increasing Ca2⫹ leak102 and
reducing SR Ca2⫹ content.
Most reports show no change in the density of ICa during
hypertrophy and failure.103 This may be due to the presence of
fewer channels, with upregulation of the remaining channels,5,104 consistent with work showing an increase in the
activity of single channels from failing human ventricular
myocytes.105 This upregulation may be due to channel phosphorylation,104 which may also explain, at least in part, the
blunted response of failing hearts to ␤-adrenergic stimulation.
NCX expression and current density appear to be increased
in hypertrophy and failure,106 although it has recently been
shown that Ca2⫹ transport via NCX is virtually unchanged in
rat ventricular myocytes after myocardial infarction if NCX
function is normalized to cell volume.107
Thus, there have been many studies of the changes that
occur in the hypertrophied and failing heart and, in particular,
of proteins that are normally found at the t-tubules. There is
also evidence that the t-tubule structure changes in these
conditions. However, there is a paucity of data regarding
whether these changes are associated with changes in the
distribution of proteins. It is tempting to speculate that
remodeling of the t-tubules and changes in Ca2⫹-handling
protein expression and distribution might alter excitationcontraction coupling in failing ventricular myocytes.
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June 13, 2003
Relevance of Changes in T-Tubule Structure and
Protein Expression to Function
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Myocytes from newborn animals have a relatively large
surface area/volume ratio,99 and myofilaments are located in
the subsarcolemmal region.84 It appears likely that sufficient
Ca2⫹ can enter via the surface membrane to activate the
myofilaments. However, these cells also show marked gradients of Ca2⫹ during excitation-contraction coupling: Ca2⫹
rises initially at the cell periphery and then diffuses into the
center of the cell.4 The development of the t-tubules is
associated with a transition to the mature pattern of
excitation-contraction coupling, which shows a synchronous
increase of [Ca2⫹] across the cell.4
The ability of ICa to trigger Ca2⫹ release from the SR is
reduced in myocytes from failing rat hearts.95 It has been
suggested that this may be due to changes in the colocalization of the L-type Ca2⫹ channel and RyR, increased separation of the t-tubule from the SR, or t-tubule remodeling. A
confounding factor is that 4 different isoforms of the ␣1C
subunit of the L-type Ca2⫹ channel are expressed in the
normal human heart,108 and isoform switching occurs in
failing human myocytes. In addition, several lines of evidence indicate hyperphosphorylation of PKA target proteins
in human heart failure,102 and because key target proteins
appear to be localized at the t-tubules (above), changes in
protein phosphorylation at the t-tubules may also underlie
changes in excitation-contraction coupling.104
However, many of these proposed mechanisms rely on a
change in the efficiency with which ICa triggers SR Ca2⫹ release.
It has been cogently argued that such a mechanism can produce
only short-lived changes in the size of the Ca2⫹ transient and that
it is more likely that a decrease in SR Ca2⫹ content underlies the
decrease in the Ca2⫹ transient.109 However, it remains possible
that a reduction in t-tubule density in heart failure5,6 could
desynchronize Ca2⫹ release on electrical stimulation,1,57 reducing peak [Ca2⫹]i and slowing its time course.110
The location of NCX is also of interest because the activity
of this protein appears to be upregulated in heart failure,
which can improve contractile function111,112 but may also
lead to arrhythmias113; however, it is unclear whether it
retains its t-tubular location during failure.
Conclusions and Unanswered Questions
It is clear that the t-tubules are not simple invaginations of the
sarcolemma. Ca2⫹-handling proteins in particular appear to be
located predominantly within the t-tubules, which therefore play
a central role in excitation-contraction coupling, functioning as
the major site for Ca2⫹ entry and release, allowing synchronous
Ca2⫹ release throughout the cell and Ca2⫹ removal from the
cytoplasm. The t-tubules may also be an important site for
modulation of contraction via NCX. What is less clear is
whether the density of these proteins is greater within the
t-tubules than at the surface membrane, which has important
implications for protein trafficking, or whether the fraction of
these proteins within the t-tubules simply reflects the fraction of
membrane within the t-tubules. Accurate estimates of protein
and membrane fraction are required, although it is notable that
despite the problems associated with immunohistochemical and
electrophysiological investigation of the t-tubules, both tech-
niques show apparent concentration of some proteins within the
t-tubules, and many ultrastructural studies agree with capacitance measurements of the amount of cell membrane (⬇30%)
located in the t-tubules.
There are, however, many other questions that have not been
fully answered: how are the t-tubules maintained, and how and
why do they change during development, hypertrophy, and heart
failure? How are the proteins concentrated at the t-tubules
targeted to this part of the cell membrane? How does protein
distribution change during development, hypertrophy, and failure, and what role does this play in the altered function observed
in these conditions? What are the electrical properties of the
t-tubules, and why are Ca2⫹-efflux pathways located close to
Ca2⫹-release sites? These and many other questions will provide
a future challenge in elucidating and understanding the role and
the importance of the t-tubules in health and disease.
Acknowledgments
The authors acknowledge financial support from the Wellcome Trust
and British Heart Foundation and thank Dr Richard Sainson for
helpful discussion and Tim Lee for the preparation of Figure 2.
References
1. Heinzel FR, Bito V, Volders PG, Antoons G, Mubagwa K, Sipido KR. Spatial
and temporal inhomogeneities during Ca2⫹ release from the sarcoplasmic
reticulum in pig ventricular myocytes. Circ Res. 2002;91:1023–1030.
2. Soeller C, Cannell MB. Examination of the transverse tubular system in
living cardiac rat myocytes by 2-photon microscopy and digital
image–processing techniques. Circ Res. 1999;84:266 –275.
3. Forbes MS, van Neil EE. Membrane systems of guinea pig myocardium:
ultrastructure and morphometric studies. Anat Rec. 1988;222:362–379.
4. Haddock PS, Coetzee WA, Cho E, Porter L, Katoh H, Bers DM, Jafri
MS, Artman M. Subcellular [Ca2⫹]i gradients during excitationcontraction coupling in newborn rabbit ventricular myocytes. Circ Res.
1999;85:415– 427.
5. He J, Conklin MW, Foell JD, Wolff MR, Haworth RA, Coronado R,
Kamp TJ. Reduction in density of transverse tubules and L-type Ca2⫹
channels in canine tachycardia-induced heart failure. Cardiovasc Res.
2001;49:298 –307.
6. Wong C, Soeller C, Burton L, Cannell MB. Changes in transversetubular system architecture in myocytes from diseased human ventricles.
Biophys J. 2002;82:a588. Abstract.
7. Bossen EH, Sommer JR, Waugh RA. Comparative stereology of the
mouse and finch left ventricle. Tissue Cell. 1978;10:773–784.
8. Bossen EH, Sommer JR. Comparative stereology of the lizard and frog
myocardium. Tissue Cell. 1984;16:173–178.
9. Ayettey AS, Navaratnam V. The T-tubule system in the specialized and
general myocardium of the rat. J Anat. 1978;127:125–140.
10. Kirk MM, Izu LT, Chen-Izu Y, McCulle SL, Wier WG, Balke CW,
Shorofsky SR. Role of the transverse-axial tubule system in generating
calcium sparks and calcium transients in rat atrial myocytes. J Physiol.
2003;547:441– 451.
11. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force.
2nd ed. Dordrecht, Netherlands: Kluwer Academic Publishers; 2001.
12. Page E. Quantitative ultrastructural analysis in cardiac membrane physiology. Am J Physiol. 1978;235:C147–C158.
13. Sommer JR, Jennings RB. Ultrastructure of Cardiac Muscle. New York,
NY: Raven Press; 1992.
14. Forbes MS, Hawkey LA, Sperelakis N. The transverse-axial tubular
system (TATS) of mouse myocardium: its morphology in the
developing and adult animal. Am J Anat. 1984;170:143–162.
15. Page E, McCallister LP, Power B. Sterological measurements of cardiac
ultrastructures implicated in excitation-contraction coupling. Proc Natl
Acad Sci U S A. 1971;68:1465–1466.
16. Blatter LA, Niggli E. Confocal near-membrane detection of calcium in
cardiac myocytes. Cell Calcium. 1998;23:269 –279.
17. Yao A, Spitzer KW, Ito N, Zaniboni M, Lorell BH, Barry WH. The
restriction of diffusion of cations at the external surface of cardiac
myocytes varies between species. Cell Calcium. 1997;22:431– 438.
Brette and Orchard
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
18. Chen F, Mottino G, Klitzner TS, Philipson KD, Frank JS. Distribution
of the Na⫹/Ca2⫹ exchange protein in developing rabbit myocytes. Am J
Physiol. 1995;268:C1126 –C1132.
19. Mitcheson JS, Hancox JC, Levi AJ. Action potentials, ion channel
currents and transverse tubule density in adult rabbit ventricular
myocytes maintained for 6 days in cell culture. Pflugers Arch. 1996;
431:814 – 827.
20. Kostin S, Scholz D, Shimada T, Maeno Y, Mollnau H, Hein S, Schaper
J. The internal and external protein scaffold of the T-tubular system in
cardiomyocytes. Cell Tissue Res. 1998;294:449 – 460.
21. Brandt N. Identification of two populations of cardiac microsomes with
nitrendipine receptors: correlation of the distribution of dihydropyridine
receptors with organelle specific markers. Arch Biochem Biophys. 1985;
242:306 –319.
22. Takagishi Y, Yasui K, Severs NJ, Murata Y. Species-specific difference
in distribution of voltage-gated L-type Ca2⫹ channels of cardiac
myocytes. Am J Physiol. 2000;279:C1963–C1969.
23. Carl SL, Felix K, Caswell AH, Brandt NR, Ball WJ Jr, Vaghy PL,
Meissner G, Ferguson DG. Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine
receptor in rabbit ventricle and atrium. J Cell Biol. 1995;129:672– 682.
24. Kawai M, Hussain M, Orchard CH. Excitation-contraction coupling in
rat ventricular myocytes after formamide-induced detubulation. Am J
Physiol. 1999;277:H603–H609.
25. Musa H, Lei M, Honjo H, Jones SA, Dobrzynski H, Lancaster MK,
Takagishi Y, Henderson Z, Kodama I, Boyett MR. Heterogeneous
expression of Ca2⫹ handling proteins in rabbit sinoatrial node. J Histochem Cytochem. 2002;50:311–324.
26. Scriven DR, Dan P, Moore ED. Distribution of proteins implicated in
excitation-contraction coupling in rat ventricular myocytes. Biophys J.
2000;79:2682–2691.
27. Doyle DD, Kamp TJ, Palfrey HC, Miller RJ, Page E. Separation of
cardiac plasmalemma into cell surface and T-tubular components: distribution of saxitoxin- and nitrendipine-binding sites. J Biol Chem.
1986;261:6556 – 6563.
28. Frank JS, Mottino G, Reid D, Molday RS, Philipson KD. Distribution of
the Na⫹-Ca2⫹ exchange protein in mammalian cardiac myocytes: an
immunofluorescence and immunocolloidal gold-labeling study. J Cell
Biol. 1992;117:337–345.
29. Kieval RS, Bloch RJ, Lindenmayer GE, Ambesi A, Lederer WJ. Immunofluorescence localization of the Na-Ca exchanger in heart cells. Am J
Physiol. 1992;263:C545–C550.
30. Petrecca K, Atanasiu R, Grinstein S, Orlowski J, Shrier A. Subcellular
localization of the Na⫹/H⫹ exchanger NHE1 in rat myocardium. Am J
Physiol. 1999;276:H709 –H717.
31. McDonough AA, Zhang Y, Shin V, Frank JS. Subcellular distribution of
sodium pump isoform subunits in mammalian cardiac myocytes. Am J
Physiol. 1996;270:C1221–C1227.
32. Maier SKG, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T,
Catterall WA. An unexpected role for brain-type sodium channels in
coupling of cell surface depolarization to contraction in the heart. Proc
Natl Acad Sci U S A. 2002;99:4073– 4078.
33. Barry DM, Trimmer JS, Merlie JP, Nerbonne JM. Differential
expression of voltage-gated K⫹ channel subunits in adult rat heart:
relation to functional K⫹ channels? Circ Res. 1995;77:361–369.
34. Takeuchi S, Takagishi Y, Yasui K, Murata Y, Toyama J, Kodama I.
Voltage-gated K⫹ channel, Kv4.2, localizes predominantly to the
transverse-axial tubular system of the rat myocyte. J Mol Cell Cardiol.
2000;32:1361–1369.
35. Jones SA, Morton MJ, Hunter M, Boyett MR. Expression of TASK-1,
a pH-sensitive twin-pore domain K⫹ channel, in rat myocytes. Am J
Physiol. 2002;283:H181–H185.
36. Clark RB, Tremblay A, Melnyk P, Allen BG, Giles WR, Fiset C.
T-tubule localization of the inward-rectifier K⫹ channel in mouse ventricular myocytes: a role in K⫹ accumulation. J Physiol. 2001;537:
979 –992.
37. Pucéat M, Korichneva I, Cassoly R, Vassort G. Identification of band
3-like proteins and Cl⫺/HCO3⫺ exchange in isolated cardiomyocytes.
J Biol Chem. 1995;270:1315–1322.
38. Jurevicius J, Fischmeister R. cAMP compartmentation is responsible for
a local activation of cardiac Ca2⫹ channels by ␤-adrenergic agonists.
Proc Natl Acad Sci U S A. 1996;93:295–299.
39. Gao T, Puri TS, Gerhardstein BL, Chien AJ, Green RD, Hosey MM.
Identification and subcellular localization of the subunits of L-type
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
Mammalian T-Tubule Function
1191
calcium channels and adenylyl cyclase in cardiac myocytes. J Biol
Chem. 1997;272:19401–19407.
Laflamme MA, Becker PL. Gs and adenylyl cyclase in transverse tubules
of heart: implications for cAMP-dependent signaling. Am J Physiol.
1999;277:H1841–H1848.
Yang J, Drazba JA, Ferguson DG, Bond M. A-kinase anchoring protein
100 (AKAP100) is localized in multiple subcellular compartments in the
adult rat heart. J Cell Biol. 1998;142:511–522.
Santana LF, Chase EG, Votaw VS, Nelson MT, Greven R. Functional
coupling of calcineurin and protein kinase A in mouse ventricular
myocytes. J Physiol. 2002;544:57– 69.
Davare MA, Avdonin V, Hall DD, Peden EM, Burette A, Weinberg RJ, Horne
MC, Hoshi T, Hell JW. A ␤2 adrenergic receptor signaling complex assembled
with the Ca2⫹ channel Cav1.2. Science. 2001;293:98–101.
Barouch LA, Harrison RW, Skaf MW, Rosas GO, Cappola TP, Kobeissi
ZA, Hobai IA, Lemmon CA, Burnett AL, O’Rourke B, Rodriguez ER,
Huang PL, Lima JA, Berkowitz DE, Hare JM. Nitric oxide regulates the
heart by spatial confinement of nitric oxide synthase isoforms. Nature.
2002;416:337–339.
Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological
implications. Physiol Rev. 1999;79:763– 854.
Lipp P, Bootman MD. The physiology and molecular biology of cardiac
Na/Ca exchange. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology
From Cell to Bedside. Philadelphia, Pa: WB Saunders Co; 2000.
Wier WG, Balke CW. Ca2⫹ release mechanisms, Ca2⫹ sparks, and local
control of excitation-contraction coupling in normal heart muscle. Circ
Res. 1999;85:770 –776.
Franzini-Armstrong C, Protasi F, Ramesh V. Comparative ultrastructure
of Ca2⫹ release units in skeletal and cardiac muscle. Ann N Y Acad Sci.
1998;853:20 –30.
Jorgensen AO, Shen AC, Daly P, MacLennan DH. Localization of
Ca2⫹⫹Mg2⫹-ATPase of the sarcoplasmic reticulum in adult rat papillary
muscle. J Cell Biol. 1982;93:883– 892.
Evans AM, Cannell MB. The role of L-type Ca2⫹ current and Na⫹ currentstimulated Na/Ca exchange in triggering SR calcium release in guinea-pig
cardiac ventricular myocytes. Cardiovasc Res. 1997;35:294–302.
Sipido KR, Maes M, Van de WF. Low efficiency of Ca2⫹ entry through
the Na⫹-Ca2⫹ exchanger as trigger for Ca2⫹ release from the sarcoplasmic reticulum: a comparison between L-type Ca2⫹ current and
reverse-mode Na⫹-Ca2⫹ exchange. Circ Res. 1997;81:1034 –1044.
Leblanc N, Hume JR. Sodium current-induced release of calcium from
cardiac sarcoplasmic reticulum. Science. 1990;248:372–376.
Trafford AW, Diaz ME, O’Neill SC, Eisner DA. Comparison of subsarcolemmal and bulk calcium concentration during spontaneous calcium release
in rat ventricular myocytes. J Physiol. 1995;488:577–586.
Cordeiro JM, Spitzer KW, Giles WR, Ershler PE, Cannell MB, Bridge
JH. Location of the initiation site of calcium transients and sparks in
rabbit heart Purkinje cells. J Physiol. 2001;531:301–314.
Huser J, Lipsius SL, Blatter LA. Calcium gradients during excitationcontraction coupling in cat atrial myocytes. J Physiol. 1996;494:641–651.
Lipp P, Huser J, Pott L, Niggli E. Spatially non-uniform Ca2⫹ signals
induced by the reduction of transverse tubules in citrate-loaded
guinea-pig ventricular myocytes in culture. J Physiol. 1996;497:
589 –597.
Brette F, Komukai K, Orchard CH. Validation of formamide as a
detubulation agent in isolated rat cardiac cells. Am J Physiol. 2002;283:
H1720 –H1728.
Yang Z, Pascarel C, Steele DS, Komukai K, Brette F, Orchard CH.
Na⫹-Ca2⫹ exchange activity is localized in the t-tubules of rat ventricular
myocytes. Circ Res. 2002;91:315–322.
Shacklock PS, Wier WG, Balke CW. Local Ca2⫹ transients (Ca2⫹
sparks) originate at transverse tubules in rat heart cells. J Physiol.
1995;487:601– 608.
Christé G. Localization of K⫹ channels in the T-tubules of cardiomyocytes as suggested by the parallel decay of membrane capacitance, IK1
and IKATP during culture and by delayed IK1 response to barium. J Mol
Cell Cardiol. 1999;31:2207–2213.
Shepherd N, McDonough HB. Ionic diffusion in transverse tubules of
cardiac ventricular myocytes. Am J Physiol. 1998;275:H852–H860.
Korchev YE, Negulyaev YA, Edwards CR, Vodyanoy I, Lab MJ. Functional localization of single active ion channels on the surface of a living
cell. Nat Cell Biol. 2000;2:616 – 619.
Komukai K, Brette F, Yamanushi TT, Orchard CH. K⫹ current distribution in rat sub-epicardial ventricular myocytes. Pflugers Arch. 2002;
444:532–538.
1192
Circulation Research
June 13, 2003
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
64. Brette F, Komukai K, Orchard CH. Importance of T-tubules in the
response of cardiac myocytes to ␤-adrenergic stimulation. Biophys J.
2002;82:a96. Abstract.
65. Huxley AF, Taylor RE. Local activation of striated muscle fibres.
J Physiol. 1958;144:426 – 441.
66. Katz MA. Physiology of the Heart. New York, NY: Raven Press; 1992.
67. Tidball JG, Smith R, Shattock MJ, Bers DM. Differences in action
potential configuration in ventricular trabeculae correlate with differences in density of transverse tubule-sarcoplasmic reticulum couplings. J Mol Cell Cardiol. 1988;20:539 –546.
68. Jack JJB, Noble D, Tsien RW. Electric Current Flow in Excitable Cells.
Oxford, UK: Oxford University Press; 1985.
69. Kim AM, Vergara JL. Supercharging accelerates T-tubule membrane
potential changes in voltage clamped frog skeletal muscle fibers.
Biophys J. 1998;75:2098 –2116.
70. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983;245:C1–C14.
71. Goldhaber JI, Lamp ST, Walter DO, Garfinkel A, Fukumoto GH, Weiss JN.
Local regulation of the threshold for calcium sparks in rat ventricular
myocytes: role of sodium-calcium exchange. J Physiol. 1999;520:431–438.
72. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events
underlying excitation-contraction coupling in heart muscle. Science.
1993;262:740 –744.
73. Tanaka H, Sekine T, Kawanishi T, Nakamura R, Shigenobu K. Intrasarcomere [Ca2⫹] gradients and their spatio-temporal relation to Ca2⫹
sparks in rat cardiomyocytes. J Physiol. 1998;508:145–152.
74. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. Direct measurement of SR release flux by tracking “Ca2⫹ spikes” in rat cardiac
myocytes. J Physiol. 1998;512:677– 691.
75. Blatter LA, Kockskamper J, Sheehan KA, Zima AV, Huser J, Lipsius
SL. Local calcium gradients during excitation-contraction coupling and
alternans in atrial myocytes. J Physiol. 2003;546:19 –31.
76. Brette F, Orchard CH. Effect of ␤-adrenergic stimulation on subcellular
Ca gradients in control and detubulated rat ventricular myocytes.
Biophys J. 2003;84:a200. Abstract.
77. Song LS, Wang SQ, Xiao RP, Spurgeon H, Lakatta EG, Cheng H.
␤-Adrenergic stimulation synchronizes intracellular Ca2⫹ release during
excitation-contraction coupling in cardiac myocytes. Circ Res. 2001;88:
794–801.
78. Levi AJ, Spitzer KW, Kohmoto O, Bridge JH. Depolarization-induced
Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac
myocytes. Am J Physiol. 1994;266:H1422–H1433.
79. Lederer WJ, Niggli E, Hadley RW. Sodium-calcium exchange in
excitable cells: fuzzy space. Science. 1990;248:283.
80. Janvier NC, Boyett MR. The role of Na-Ca exchange current in the
cardiac action potential. Cardiovasc Res. 1996;32:69 – 84.
81. Zaccolo M, Pozzan T. Discrete microdomains with high concentration
of cAMP in stimulated rat neonatal cardiac myocytes. Science. 2002;
295:1711–1715.
82. Xiao RP. ␤-Adrenergic signaling in the heart: dual coupling of the
␤2-adrenergic receptor to Gs and Gi proteins. Sci STKE. 2001;
2001:RE15.
83. Brette F, Rodriguez P, Coyler J, Orchard CH. Effect of detubulation on
phospholamban phosphorylation in rat ventricular myocytes. J Physiol.
2002;544P:46P. Abstract.
84. Smolich JJ. Ultrastructural and functional features of the developing mammalian heart: a brief overview. Reprod Fertil Dev. 1995;7:451–461.
85. Lee E, Marcucci M, Daniell L, Pypaert M, Weisz OA, Ochoa GC,
Farsad K, Wenk MR, De Camilli P. Amphiphysin 2 (Bin1) and T-tubule
biogenesis in muscle. Science. 2002;297:1193–1196.
86. Carozzi AJ, Ikonen E, Lindsay MR, Parton RG. Role of cholesterol in
developing T-tubules: analogous mechanisms for T-tubule and caveolae
biogenesis. Traffic. 2000;1:326 –341.
87. Razani B, Woodman SE, Lisanti MP. Caveolae: from cell biology to
animal physiology. Pharmacol Rev. 2002;54:431– 467.
88. Franzini-Armstrong C. Simultaneous maturation of transverse tubules
and sarcoplasmic reticulum during muscle differentiation in the mouse.
Dev Biol. 1991;146:353–363.
89. Ishikawa H. Formation of elaborate networks of T-system tubules in
cultured skeletal muscle with special reference to the T-system formation. J Cell Biol. 1968;38:51– 66.
90. Page E, McCallister LP. Quantitative electron microscopic description
of heart muscle cells: application to normal, hypertrophied and thyroxinstimulated hearts. Am J Cardiol. 1973;31:172–181.
91. Keung EC, Toll L, Ellis M, Jensen RA. L-type cardiac calcium channels
in doxorubicin cardiomyopathy in rats morphological, biochemical, and
functional correlations. J Clin Invest. 1991;87:2108 –2113.
92. Schaper J, Froede R, Hein S, Buck A, Hashizume H, Speiser B, Friedl A,
Bleese N. Impairment of the myocardial ultrastructure and changes of the
cytoskeleton in dilated cardiomyopathy. Circulation. 1991;83:504–514.
93. Kaprielian RR, Stevenson S, Rothery SM, Cullen MJ, Severs NJ.
Distinct patterns of dystrophin organization in myocyte sarcolemma and
transverse tubules of normal and diseased human myocardium. Circulation. 2000;101:2586 –2594.
94. Ohler A, Houser SR, Tomaselli GF, Rourke B. Transverse tubules are
unchanged in myocytes from failing human heart. Biophys J. 2002;
82:a590. Abstract.
95. Gomez AM, Valdivia HH, Cheng H, Lederer MR, Santana LF, Cannell
MB, McCune SA, Altschuld RA, Lederer WJ. Defective excitationcontraction coupling in experimental cardiac hypertrophy and heart
failure. Science. 1997;276:800 – 806.
96. Gomez AM, Guatimosim S, Dilly KW, Vassort G, Lederer WJ. Heart
failure after myocardial infarction: altered excitation-contraction
coupling. Circulation. 2001;104:688 – 693.
97. Page E, Buecker JL. Development of dyadic junctional complexes between
sarcoplasmic reticulum and plasmalemma in rabbit left ventricular myocardial cells: morphometric analysis. Circ Res. 1981;48:519–522.
98. Cohen NM, Lederer WJ. Changes in the calcium current of rat heart
ventricular myocytes during development. J Physiol. 1988;406:115–146.
99. Haddock PS, Coetzee WA, Artman M. Na⫹/Ca2⫹ exchange current and
contractions measured under Cl⫺-free conditions in developing rabbit
hearts. Am J Physiol. 1997;273:H837–H846.
100. Bers DM, Philipson KD, Langer GA. Cardiac contractility and sarcolemmal calcium binding in several cardiac muscle preparations. Am J
Physiol. 1981;240:H576 –H583.
101. Tomaselli GF, Marban E. Electrophysiological remodeling in hypertrophy and heart failure. Cardiovasc Res. 1999;42:270 –283.
102. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit
N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the
calcium release channel (ryanodine receptor): defective regulation in
failing hearts. Cell. 2000;101:365–376.
103. Benitah JP, Gomez AM, Fauconnier J, Kerfant BG, Perrier E, Vassort G,
Richard S. Voltage-gated Ca2⫹ currents in the human pathophysiologic
heart: a review. Basic Res Cardiol. 2002;97(suppl 1):I-11–I-18.
104. Chen X, Piacentino V III, Furukawa S, Goldman B, Margulies KB,
Houser SR. L-type Ca2⫹ channel density and regulation are altered in
failing human ventricular myocytes and recover after support with
mechanical assist devices. Circ Res. 2002;91:517–524.
105. Schroder F, Handrock R, Beuckelmann DJ, Hirt S, Hullin R, Priebe L,
Schwinger RHG, Weil J, Herzig S. Increased availability and open
probability of single L-type calcium channels from failing compared
with nonfailing human ventricle. Circulation. 1998;98:969 –976.
106. Barry WH. Na⫹-Ca2⫹ exchange in failing myocardium: friend or foe?
Circ Res. 2000;87:529 –531.
107. Gomez AM, Schwaller B, Porzig H, Vassort G, Niggli E, Egger M.
Increased exchange current but normal Ca2⫹ transport via Na⫹-Ca2⫹
exchange during cardiac hypertrophy after myocardial infarction. Circ
Res. 2002;91:323–330.
108. Yang Y, Chen X, Margulies K, Jeevanandam V, Pollack P, Bailey BA, Houser
SR. L-type Ca2⫹ channel ␣1c subunit isoform switching in failing human
ventricular myocardium. J Mol Cell Cardiol. 2000;32:973–984.
109. Eisner DA, Trafford AW. Heart failure and the ryanodine receptor: does
Occam’s razor rule? Circ Res. 2002;91:979 –981.
110. Litwin SE, Zhang D, Bridge JH. Dyssynchronous Ca2⫹ sparks in
myocytes from infarcted hearts. Circ Res. 2000;87:1040 –1047.
111. Mattiello JA, Margulies KB, Jeevanandam V, Houser SR. Contribution
of reverse-mode sodium-calcium exchange to contractions in failing
human left ventricular myocytes. Cardiovasc Res. 1998;37:424 – 431.
112. Sipido KR, Volders PG, De Groot SH, Verdonck F, Van de Werf F,
Wellens HJ, Vos MA. Enhanced Ca2⫹ release and Na/Ca exchange
activity in hypertrophied canine ventricular myocytes: potential link
between contractile adaptation and arrhythmogenesis. Circulation.
2000;102:2137–2144.
113. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodiumcalcium exchange, inward rectifier potassium current, and residual
␤-adrenergic responsiveness. Circ Res. 2001;88:1159 –1167.
T-Tubule Function in Mammalian Cardiac Myocytes
Fabien Brette and Clive Orchard
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Circ Res. 2003;92:1182-1192
doi: 10.1161/01.RES.0000074908.17214.FD
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