2017 PHYS REV Túbulo T Cardíaco Microanatomia e Função

Physiol Rev 97: 227–252, 2017
Published November 23, 2016; doi:10.1152/physrev.00037.2015
CARDIAC T-TUBULE MICROANATOMY

AND FUNCTION
TingTing Hong and Robin M. Shaw
Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California; and Department of Medicine,
University of California Los Angeles, Los Angeles, California
Hong T, Shaw RM. Cardiac T-Tubule Microanatomy and Function. Physiol Rev 97: 227–
L
252, 2017. Published November 23, 2016; doi:10.1152/physrev.00037.2015.—
Unique to striated muscle cells, transverse tubules (t-tubules) are membrane organ-
elles that consist of sarcolemma penetrating into the myocyte interior, forming a highly
branched and interconnected network. Mature t-tubule networks are found in mam-
malian ventricular cardiomyocytes, with the transverse components of t-tubules occurring near
sarcomeric z-discs. Cardiac t-tubules contain membrane microdomains enriched with ion channels
and signaling molecules. The microdomains serve as key signaling hubs in regulation of cardiomy-
ocyte function. Dyad microdomains formed at the junctional contact between t-tubule membrane
and neighboring sarcoplasmic reticulum are critical in calcium signaling and excitation-contraction
coupling necessary for beat-to-beat heart contraction. In this review, we provide an overview of the
current knowledge in gross morphology and structure, membrane and protein composition, and
function of the cardiac t-tubule network. We also review in detail current knowledge on the
formation of functional membrane subdomains within t-tubules, with a particular focus on the
cardiac dyad microdomain. Lastly, we discuss the dynamic nature of t-tubules including membrane
turnover, trafficking of transmembrane proteins, and the life cycles of membrane subdomains such
as the cardiac BIN1-microdomain, as well as t-tubule remodeling and alteration in diseased hearts.
Understanding cardiac t-tubule biology in normal and failing hearts is providing novel diagnostic and
therapeutic opportunities to better treat patients with failing hearts.
I. INTRODUCTION 227 partmentalize transmembrane ion handling proteins and

II. CARDIAC T-TUBULE STRUCTURE 228 signaling molecules. Together, t-tubules are effective organ-
III. TRANSMEMBRANE PROTEINS AT... 236 elles that serve as a centralized signaling hub controlling
IV. FUNCTION OF CARDIAC T-TUBULES 238 cardiac contractile function and electrophysiology.
V. LIFE CYCLE OF CARDIAC T-TUBULES 242
VI. ALTERATION OF T-TUBULE... 244 The most recognized function of t-tubules is regulation of
VII. CONCLUSIONS 246 cardiac EC coupling by concentrating voltage-gated L-type
calcium channels (LTCCs) and positioning them in close
proximity to calcium sense and release channels, ryanodine
I. INTRODUCTION
receptors (RyRs), at the junctional membrane of sarcoplas-
mic reticulum (jSR). The closely approximated LTCCs and
Cardiac transverse tubules (t-tubules) are highly branched
RyRs form calcium releasing units, dyads, where calcium
invaginations of cardiomyocyte sarcolemma that are rich in
transients are initiated following each beat-to-beat action
ion channels important for excitation-contraction (EC)
potential. With recent advances in high-resolution imaging
coupling, maintenance of resting membrane potential, ac-
tion potential initiation and regulation, and signaling trans- technologies, detailed t-tubule microdomain structural or-
duction. Specific to striated muscle cells, t-tubules are in- ganization and newly recognized functions are starting to
vaginations of the sarcolemma, penetrating into the intra- emerge. For instance, the t-tubular invagination is not
cellular space of myocytes. These tubular invaginations smooth, but consists of extensive membrane microfolds
form a complex network with transverse tubules that are sculpted by a cardiac isoform of a BAR domain containing
interconnected within the cytoplasm by longitudinal tu- protein bridging integrator 1 (BIN1 or amphiphysin 2) (91).
bules. The transverse tubules occur around myofilaments, Cardiac BIN1 (cBIN1) organized membrane microfolds
anchoring to sarcomeric z-discs through costameres. The generate diffusion barriers which slow t-tubule-associated
phospholipid-rich t-tubule lipid bilayers are shaped and extracellular ionic diffusion, preserving electrical stability
maintained by membrane scaffolding proteins and intracel- of myocytes during tachycardia-related fluctuations of ionic
lular cytoskeleton, as well as surrounding extracellular ma- concentrations in the local extracellular and possibly intra-
trix. T-tubule membrane contains microdomains that com- cellular environment (91). In addition, cBIN1-microfolds
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TINGTING HONG AND ROBIN M. SHAW
also organize local microdomains for efficient and dynamic transverse to the long axis of myocytes and occurs peri-
regulation of cardiac dyad function, regulating EC coupling odically at sarcomeric z-discs of myofilament (126). By
(66, 93, 94). using transmission electron microscopy (TEM) imaging
methods in 1957, Lindner (126) identified that these
Cardiac t-tubules are also substantially remodeled during transverse cardiac t-tubules are open to extracellular
heart failure (38, 52, 89, 97, 98, 108, 130-134, 180, 202, space. The longitudinal axial component of t-tubules was
207, 208, 220, 224, 230). In addition to the observed loss identified later in 1970s (201), adding the complexity of
and gross diminishment of the t-tubule network, protein the t-tubule system. The 1960s and 1970s was a period of
components at t-tubules including ion channels and signal- improved TEM and sample preparation technology
ing molecules are reported to be either reduced at the t-tu- which led to prolific t-tubule morphology research (60,
bule surface or redistributed elsewhere to non-t-tubule sar- 62, 153, 190, 191, 201), revealing the organization of
colemma (20, 93, 134). Loss of complex t-tubules in heart t-tubule networks (see details in sect. IIB). The discovery
failure not only causes impaired contractile function due to of dyads and calcium releasing units at t-tubules in late
disrupted dyads and the resultant EC uncoupling, but also 1980s and early 1990s (31, 59) led to function-driven
alters local concentration gradients and increases suscepti- research of cardiac t-tubules. In the past 5 years, with
bility to ventricular arrhythmia (66, 85, 91, 157, 163, 179, advance in super-resolution fluorescent microscopy and
181, 213). The molecular and cellular mechanisms of t-tu- three-dimensional TEM tomography reconstruction, di-
bule remodeling in failing cardiomyocytes, in particular the rect visualization into membrane ultrastructure and pro-
alterations in t-tubule membrane ultrastructure and sub- tein localization has provided unprecedented details of
domains, is an active area of interest among cardiac biolo- membrane microdomains within cardiac t-tubules, which
gists. will be discussed in more detail in section IIC.
This review summarizes and cites the current knowledge of Present studies of cardiac t-tubules include myocytes
cardiac t-tubule morphology, structure, components, and from the atria (109), ventricles, and conducting system
physiological functions, with emphasis on the organization (3) and are performed across different species including
and function of microdomains within t-tubules. The dy- small murine, large mammals, and human hearts (20).
namic life cycle of t-tubule membrane and protein compo- Cardiac t-tubules have classic features that distinguish them
nents, as well as the current understanding of microdomain
from skeletal t-tubules. It is well documented that t-tubules
remodeling in heart failure, will also be discussed.
of some form exist in all cardiomyocytes and that the t-tu-
bule system in mammalian ventricular cardiomyocytes is
II. CARDIAC T-TUBULE STRUCTURE the most extensive (see FIGURE 1 for a schematic illustra-
tion of ventricular cardiomyocyte organziation and t-tu-
bule membrane structure occurring near z-discs). Re-
A. History of T-Tubule Structure-Related cently identified t-tubule membrane subdomains also
Research provide a structural foundation and clearer understand-
ing of t-tubule regulation of cardiomyocyte function.
Striated muscle cells have a unique membrane system, the This section reviews the gross morphology, membrane
transverse-tubules (t-tubules), or more precisely, the trans- ultrastructure and microdomains, and unique features of
verse-axial-tubular system. T-tubules are sarcolemma in- cardiac t-tubules.
vaginations formed by membrane tubules that are primarily
perpendicular to myocyte longitudinal edges. These mem-
B. Gross Morphology and Geometry
brane tubular structures are interconnected in a complex
network. Retzius first proposed the existence of t-tubules
over 130 years ago, in 1881, when exploring the quick Early TEM images in glutaraldehyde-fixed cardiac tissues
inward spread of action potentials penetrating into muscle infiltrated with tracers such as ferritin, lanthanum salts, or
cells (see the 1967 Croonian lecture by Huxley) (95). The horseradish peroxidase identified some of the key features
first visual evidence of tubular structures around myofila- of cardiac t-tubule system (60, 62, 153, 190, 191, 201)
ment was in 1897 by light microscopy in Nystrom’s study including 1) t-tubules are continuous extension of sarco-
(95), when he injected mammalian heart muscle with India lemma with openings clearly connected to extracellular
ink to track extracellular space. Later, in 1924, Tiegs visu- space; 2) orientation of t-tubules indicates these membrane
alized Z-lines as a spiral structures where inward spread of tubules are primarily transverse and perpendicular to myo-
action potential occurs (95). However, it was not until the cyte longitudinal edges, with an axial portion running in
1950s that Huxley and his peers reported, in various parallel between myofilaments connecting the transverse
muscle cells, the structure for inward spread as tubular tubules; and 3) t-tubules wrap around the z-disc and form
membrane structures at the I band (which spans the Z- tubular structures with the flat ends of sarcoplasmic reticu-
line) (96). The primary element of cardiac t-tubules is lum known as terminal cisternae.
228 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

T-TUBULE MICRODOMAINS
Intercalated T-tubule Sarcoplasmic reticulum Sarcomere Z-disk

disc
Nucleus
Nucleus
Microtubule Golgi Endoplasmic reticulum Mitochondrion

(rough ER)
FIGURE 1. Schematic illustration of the internal structures of an adult ventricular cardiomyocyte. T-tubules,
which are enriched with voltage-gated L-type calcium channels, are positioned closely near the sarcoplasmic
reticulum, the primary internal calcium store. Sarcomeres form myofibrils, which are responsible for cardio-
myocyte contraction upon calcium release. The Golgi apparatus and microtubules serve as the “loading dock”
and “highways,” respectively, to deliver ion channels to specific subdomains on the plasma membrane.
Mitochondria provide the energy needed for the contraction of cardiomyocytes. Intercalated discs located at
the longitudinal sides of each ventricular cardiomyocyte mediate the cell-to-cell propagation of action potentials.
Since the 1990s, fast development of modern fluorescent from 21 to 64% with an average around 30%) (159, 161–
light microscopy techniques has made it feasible to image 163, 198).
cardiac t-tubules in intact cardiac tissue and live cardiomy-
ocytes. Consistent with the earlier TEM findings, two-pho- The advent of super-resolution fluorescent microscopy im-
ton and confocal images of membrane fluorescent dye-la- aging techniques can, in theory, improve XY spatial reso-
beled t-tubules reveal that these membrane invaginations lution from 250 nm with confocal microscopy to 10 –20
occur orderly at every ⬃1.8- to 2-␮m intervals perpendicu- nm, while retaining robust protein localization (227). In a
lar to the long axis of cardiomyocytes, which forms a radial study using stimulated emission depletion (STED) imaging,
“spokelike” organization in transverse section of myofila- t-tubule structure and lumen are visualized in mouse cardi-
ment near Z-disc regions (198). Three-dimensional (3D) omyocytes with new structural details (220). In addition to
volume reconstruction of stepper motor acquired z-stacks the fluorescent microscopy, scanning ion conductance mi-
of two-dimensional XY frame images acquired at incremen- croscopy (SICM) has also been used to image live cardio-
tal depth provide spatial visualization of the t-tubule gross myocyte surface topology, providing visualization of open-
morphology and network, allowing global analysis of t-tu- ings of transverse tubular invaginations (77, 154). Mean-
bule localization, size, and branching points. On the basis while, the new two-axis tilted electron microscopy imaging
of studies using these imaging techniques together with elec- in combination with three-dimensional tomography recon-
trophysiological tools, current knowledge of t-tubule mor- struction also provides new TEM features of t-tubule ultra-
phology and geometry includes the ratio between transverse structure, including the identification of variable t-tubule
and axial elements (60 vs. 40%) (198), luminal diameter luminal diameter (87) with dilation near junction (228),
(ranges from 20 to 450 nm with an average of 200 –300 nm ryanodine receptor feet at the dyad junctional membrane
for rat cardiomyocytes), average length between branching (43, 87), and membrane microfolds (91) (see more details in
points (⬃6.7 ␮m), total volume percent of cardiomyocyte sect. IIC). The use of super-resolution microscopy helps
(varies between 0.8% in mouse and 3.6% in rat cardiomy- identify LTCC and RyR localization within these micro-
ocytes), and percentage of membrane capacitance (varies folds (66).
Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org 229

C. Membrane Ultrastructure and lar signaling. In this section, we discuss main membrane
Microdomains microdomains within t-tubule membrane invaginations.
The meshlike cardiac t-tubule network with interconnected 1. cBIN1 microfolds: microdomains supporting
transverse and longitudinal tubules is now increasingly ap- LTCC-RyR dyads
preciated as a nonuniform tubular network consisting of
variable lumen diameters, differential membrane ultra- Recently, a t-tubule subdomain was found to consist of
structure, and multiple types of frequently occurring submembrane microfolds sculpted by cBIN1. With the use of
domains. These membrane ultrastructure and subdomains 3D confocal and TEM imaging, it was identified that t-tu-
not only add spatial complexity to t-tubule lumen, but also bule invaginations are actually not formed by simply
provide structural foundation necessary to fulfill the func- straight and smooth membrane on the sides, but rather with
tional needs of t-tubules in regulation of EC coupling, ex- complex and layered membrane microfolds (91). As shown
tracellular ion diffusion, membrane excitability, and cellu- in FIGURE 2A, low-magnification 2D TEM image (left) in-
A TEM
B TEM
C TEM D STED E STORM
FIGURE 2. Images of t-tubule microfolds. A: TEM images reproduced from Hong et al. (91). B: TEM images
reproduced from Forssmann and Girardier (62). C: TEM image reproduced from Lavorato et al. (117), with
permission from Springer. D: STED images reproduced from Wagner et al. (220). E: STORM images
reproduced from Jayasinghe et al. (101).

dicates that t-tubules are contoured tubules with calcium- system and then, while the heart is still beating, the myo-
dependent electron-dense precipitations along tubular cytes should be fixed in calcium-containing buffer intro-
membrane. High-magnification 2D TEM images and 3D duced via the perfusion needle securing the aorta direct-
tomography (FIGURE 2A, right panels) of transverse and ing flow into the coronaries and retrograde through the
axial cross sections of cardiac t-tubules reveal that redun- heart. The variation in sample preparation and imaging
dant membrane layers and microfolds exist along tubular techniques can possibly explain the time it has taken to
membrane (91). While it is intriguing to some readers that fully appreciate the complexity of microfolds within t-tu-
such dramatic membrane structural feature received sparse bule invaginations.
attention, reports of contoured t-tubules in mammalian car-
diomyocytes have been reported for decades. As early as in Sculpting of microfolds within cardiac t-tubules is depen-
1970, Forssmann and Girardier (62) found in rat cardiomy- dent on BIN1, which is a phospholipid-adherent membrane
ocytes that t-tubules are extensively tortuous with hairpin- scaffolding protein. BIN1, alternatively known as am-
like bends (FIGURE 2B). Following this initial study, con- phiphysin 2, is a member of the BIN1-Amphiphysin-Rvs
toured cardiac t-tubules with extra membrane layers and (BAR) domain containing protein superfamily, which con-
microfolds can be identified in multiple cardiac t-tubule sists of membrane deformation proteins involved in multi-
studies including a recent study from Franzini-Armstrong’s ple cellular processes across different tissues and organs.
group in 2015 (FIGURE 2C) (117), as well as the apparent BIN1, and in general the BAR domain protein superfamily,
variable luminal areas within t-tubules and extended mem- contributes to membrane trafficking and remodeling, cyto-
brane subdomains by super-resolution fluorescent imaging skeleton dynamics, DNA repair, cell cycle progression, and
with STED (FIGURE 2D) (220) and stochastic reconstructive apoptosis (173). With 49% sequence homology (64), BIN1
optical reconstruction microscopy (STORM) (FIGURE 2E) and its closest cousin amphiphysin 1 both belong to the
(101). By inspection, cBIN1 generated microfolds are more N-BAR subfamily containing an NH2-terminal BAR do-
concentrated at the neck of t-tubules close to tubular open- main. One of the most well-studied functions of am-
ings at the sarcolemma (FIGURE 1E in Ref. 91). Removal of phiphysins is their involvement in membrane trafficking
these microfolds in cardiac conditional Bin1-deleted adult and endocytosis (9). Interestingly, BIN1 was first identified
mouse ventricular cardiomyocytes not only increases t-tu- as a MYC oncoprotein interacting protein (36, 47), which
bule luminal diameter, but also increases the visibility of serves as a tumor suppressor by inhibiting MYC function
t-tubule openings via SICM imaging of cardiomyocyte sur- (221). Follow-up studies identified that more than 10 BIN1
face topology (91). While increasing evidence in support protein isoforms are encoded by a single gene with 20 exons
of the existence of microfolds within t-tubules starts to (FIGURE 3). All the BIN1 protein isoforms contain a con-
emerge, in our experience their fragility places an empha- served NH2-terminal lipid binding BAR domain encoded
sis on cardiomyocyte preservation protocols for electron by exons 1–9 (exon 7 is skipped outside of the neuronal
microscopy. We have found that, for TEM imaging (FIG- system), an alternatively spliced linkage coiled-coil re-
URE 2A), freshly extracted whole mouse hearts need to be gion, the MYC binding domain encoded by a ubiqui-
maintained with immediate perfusion on a Langendorff tously alternatively spliced exon 17, a constitutive exon
BAR PI CLAP MBD SH3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 14 16 17 18 19 20
N BAR 18 SH3
Ubiquitous FIGURE 3. Bin1 splice variants in adult mouse
N BAR 17-18 SH3 cardiomyocytes. Cartoon of Bin1 exons and the
splice variants found in adult mouse cardiomyo-
cytes, as well as brain and skeletal muscle. BAR,
N BAR 13-16 18 SH3 Bin-amphiphysin-Rvs domain; PI, phosphoinosi-
Brain
tide binding domain; CLAP, clathrin/AP2 bind-
N BAR 13-16 17-18 SH3
ing region; MBD, myc binding domain; SH3,
SH3 domain. For details, see Reference 89.
N BAR 13 18 SH3
Cardiac
N BAR 13 17-18 SH3
N BAR 11 18 SH3
Skeletal
N BAR 11 17-18 SH3

18, and a conserved COOH-terminal exons 19 –20 en- BIN1 in organizing microdomains in skeletal t-tubules
coded Src homology 3 (SH3) domain critical for interac- (194).
tion with cytoskeleton and other intracellular protein
partners. The middle linkage region is heavily alterna- Together with the previously identified ability of BIN1 in
tively spliced with tissue and organ specificity including a localizing and clustering LTCCs to cardiac t-tubules, these
skeletal specific exon 11 encoding the phosphoinositide cardiac BIN1⫹13⫹17 created microfolds are likely the
(PI) binding domain, a cardiac alternatively spliced exon membrane microdomains supporting cardiac dyad (LTCC-
13 encoding proline-rich domains, and neuronal co- RyR) formation. The role of BIN1 in facilitating microtu-
spliced exons 13–16 encoding a clathrin and AP2 binding bule-dependent targeted delivery of CaV1.2 channels to t-
domain (CLAP) (22, 72). tubule membrane (94) will be discussed in section III. Con-
focal imaging and biochemical results identified that
Cardiac t-tubule membrane microfold formation requires the Cav1.2 is localized to BIN1 molecules at cardiac t-tubules.
cardiac isoform BIN1⫹13⫹17 with inclusion of both the car- BIN1 decreases occur in human (93) and animal models of
diac alternatively spliced exon 13 and the ubiquitously alter- heart failure (23, 134), which affects LTCC surface expres-
natively spliced exon 17 (91, 222) (FIGURE 3). The ability of sion at the t-tubules of failing cardiomyocytes. New super-
BIN1⫹13⫹17 to induce microfolds is likely through its resolution STORM imaging further identified that BIN1-
NH2-terminal BAR domain together with the proline-rich microfolds are not only enriched with Cav1.2 channel clus-
domains encoded by cardiac exon 13 because the NH2- ters but also attract RyRs from the jSR membrane (see
terminal BAR domains oligomerize into concave-shaped supplemental movie on the Physiological Reviews website),
surfaces with positively charged patches crucial for interac- further supporting the role of BIN1 in organizing cardiac
tions with membrane lipids. Through these interactions dyads (65, 66). Thus BIN1-induced t-tubule microfolds cre-
membrane is sculpted around BIN1 organized curvature. ate local microdomains bringing sarcolemmal LTCCs to
The crystal structure of the amphiphysin N-BAR domain couple with RyRs from jSR membrane. BIN1-regulated
reveals a dimeric interaction between helixes from each coupling of LTCCs to RyRs also helps explain previous
monomer to form a 6-helix bundle (169). With the elon- observed allosteric activation of RyR following calcium
gated banana shape of the dimers (169) and the general channel activation independent of Ca2⫹ influx (73). In sum-
mary, BIN1 microdomains provide a physical understand-
function in membrane curvature formation, BIN1 has thus
ing of how cardiac calcium releasing units are organized
been referred to as the “banana” molecule (28, 79, 174).
(FIGURE 4). More detailed discussion into the multimodal
The interface between the monomers is largely hydropho-
role of BIN1 in formation of LTCC-RyR dyads and extra-
bic, and the curvature of the dimer results from how the
cellular ion diffusion will be further discussed in sections IV
monomers intersect at highly conserved kinks (28, 225) at
and V.
the end of the 6-helix bundle. These kinks are dependent on
proline residues, isomerization of which represents a rate-
2. Caveoli (caveolae): ␤-AR/LTCC signaling hubs
limiting step in dimer formation (79). Domains immedi-
ately following the N-BAR in BIN1 can further strengthen The other well-identified membrane subdomains are
its binding affinity to phospholipids for curvature forma- caveoli, which are flask-shaped membrane invaginations
tion. For instance, cardiac isoform BIN1⫹13⫹17 contains with diameter typically around 90 nm (123). In muscle cells,
proline-rich domains encoded by the cardiac spliced exon caveoli are detergent-resistant membrane structures orga-
13. These extra proline residues likely create additional nized by the plasma membrane binding protein caveolin-3,
kinks in BIN1⫹13⫹17 dimers, increasing concavity of the rich in cholesterol and sphingomyelin. Caveoli are found to
surface of the banana molecules needed for microfold for- be attached to both general sarcolemma (0.04 ␮m2 mem-
mation within t-tubules. On the other hand, inclusion of brane area/␮m3 cell volume) and t-tubule membrane (0.03
exon 17 can further separate exon 13-encoded proline-rich ␮m2 membrane area /␮m3 cell volume) in cardiomyocytes
domains from the NH2-terminal SH3 domain, preventing (159), increasing total plasma membrane area by 14 –21%
intramolecular binding between the two domains as ob- (159). Within t-tubules of cardiomyocytes, caveoli cluster
served in BIN1⫹13. As a result, the BIN1⫹13⫹17 isoform at necks of the t-tubules in the subsarcolemmal space
is particularly well suited to promote N-WASP (neuronal (151, 238). Attachment of caveolae to t-tubules not only
Wiskott-Aldrich syndrome protein)-dependent actin po- increases the spatial complexity of t-tubule lumen, but
lymerization to maintain and stabilize t-tubule membrane also differentially concentrates membrane ion channels
microfolds to sarcomeric z-discs (91). Interestingly, the and signaling molecules for localized regulation at these
skeletal muscle specific splicing of exon 11 encodes highly membrane subdomains. In cardiomyocytes, caveolae can
positively charged amino acids, increasing skeletal BIN1- house ion channels HCN4, Cav1.2, Kv1.5, Kir6.2/Sur2a,
BAR’s binding affinity to phospholipids. A recent study of Nav1.5, and NCX1, and signaling molecules such as
exon 11 skipping in zebrafish skeletal muscle reported a ␤-adrenergic receptors (␤-AR). For example, caveolin-3
dilated t-tubule pattern similar to the cardiac phenotype, is critical in concentrating ␤2-AR-cAMP signaling to the
indicating a similar function of exon 11 containing skeletal t-tubules (229). A subpopulation of LTCCs at caveolae,

Extracellular space Extracellular ions
Actin
Sarcoplasmic reticulum
RyR
Cytoplasm
BIN1
LTCC
Microtubule +Tip protein
T-tubule Sarcoplasmic reticulum Myofilament
Nucleus
Adult ventricular cardiomyocyte
FIGURE 4. Schematic illustration of BIN1-microdomain organization within cardiac t-tubules. Top: the “ba-
nana”-shaped molecule cBIN1 regulates cardiac t-tubule function through: 1) facilitating microtubule-depen-
dent forward trafficking of Cav1.2 channels (LTCC, L-type calcium channels) to cBIN1 organized membrane
microfolds at t-tubules (targeted delivery); 2) clustering of LTCCs and RyRs at cBIN1-microfolds based
microdomains; 3) creating extracellular ion slow diffusion zone within t-tubule lumen; 4) organizing microdo-
mains for dyad formation and regulation by ␤-adrenergic signaling. Bottom: cartoon of mammalian adult
ventricular cardiomyocytes, which are rod-shaped striated muscle cells with t-tubule invaginations occurring
periodically near z-discs of myofilaments.
which is under the regulation of ␤2-AR stimulation (14), teins with binding affinity to plasma membrane lipids
is reported to be important in hypertrophic signaling but particularly cholesterol and sphingomyelin. There are
not muscle contraction (137). Thus caveolae have a crit- three caveolin isoforms, caveolin-1, caveolin-2, and
ical role in myocyte function by creating microdomains caveolin-3, with caveolin-3 specific to the muscle cells
with compartmentalized specific lipid species, membrane (200). Caveolin-3 has been indicated in muscle t-tubule
proteins, and signaling molecules, facilitating functional development and is associated with developing skeletal
regulation. t-tubules, while knockout of cavolin-3 can induce abnor-
mal t-tubules in skeletal muscle (70). Furthermore, as
The membrane scaffolding proteins responsible for cave- caveolin-3 organized caveloae is critical in compartmen-
olae formation are caveolins, which are a family of pro- talized regulation of ion channels, mutations in caveo-

lin-3 are associated with channelopathies including long D. Peri-Membrane Cytoskeleton Scaffolds at
QT syndrome (5). Cardiac T-Tubules
3. Ankyrin B microdomains: NCX/NKA/InsP3 T-tubule membrane is supported by a unique set of peri-

macromolecular complex membrane scaffolds formed by both extracellular cell adhe-
sion molecules and intracellular membrane scaffolding pro-
teins tethered through cortical cytoskeleton to costamere
In addition to BIN1-microfolds and caveoli, a third set of
and sarcomere structural proteins. Different from skeletal
microdomains organized by the membrane scaffolding protein
t-tubules, cardiac t-tubule membrane is anchored to the
ankyrin B has been identified to be essential in the formation of
Z-discs of sarcomeres. In this section, peri-t-tubule mem-
the macromolecular complex of sodium calcium exchanger
brane scaffolds and cytoskeleton as well as the t-tubule
(NCX), sodium-potassium-ATPase (NKA), and inositol tris-
membrane anchors, including F-actin/N-WASP/tropomy-
phosphate receptor (InsP3) (147). Distinct from the dyad
sin, costamere dystrophin, and t-tubule membrane-associ-
macromolecular complex, the NCX/NKA/InsP3 complex is
ated dysferlin, will be discussed.
critical in removing calcium from the luminal clefts between
the t-tubule and jSR membrane, facilitating calcium decline 1. Actin filaments, tropomyosin, and N-WASP
during muscle relaxation (147). Therefore, by balancing
calcium entry through LTCC-RyR dyads and calcium re- Actin filaments have been indicated in the organization and
moval through NCX anchored at ankyrin B-microdomains, maintenance of t-tubule structure and function. Stabiliza-
intracellular calcium equilibrium can be achieved to main- tion of cardiac actin filament by cytochalasin D preserves
tain coordinated beat-to-beat heart contraction. t-tubules in rodent ventricular cardiomyocytes during ex-
tended in vitro culture, which normally loses t-tubule in-
The ankyrin B membrane microdomains are formed by vaginations (91, 118, 214). Actin depolymerization by la-
ankyrin B protein, which belongs to a family of intracel- trunculin A, on the other hand, disrupts membrane micro-
lular proteins with binding affinity to actin and spectrins. folds at t-tubules (91). The integrity of actin filaments is
Three separate genes ANK1, ANK2, and ANK3 encode heavily regulated by many actin-regulating proteins includ-
ankyrin R, ankyrin B, and ankyrin G, respectively, that ing G-actin monomer binding proteins, nucleating proteins,
are ubiquitously expressed with multiple alternatively depolymerizing proteins, capping proteins, and polymeriz-
spliced forms of each ankyrin. However, cardiomyocytes ing proteins. These regulatory proteins are important in the
express three ankyrin B isoforms including the ankyrin organization and function of the myocyte contractile appa-
B-220 kDa and two recently identified isoforms ankyrin ratus, cardiac sarcomeres that are primarily composed of
B-188 kDa and ankyrin B-212 kDa (231), and one 190- cardiac actin and myosin. For example, muscle specific tro-
kDa ankyrin G, with distinct functions in organizing pomyosin isoforms are associated with sarcomeric actins to
NCX/NKA/InsP3 complex at t-tubules or trafficking form thin filaments, responsible for myocyte contraction
Nav1.5 to intercalated discs, respectively [see more de- following intracellular calcium elevation. Interestingly, in
tails in reviews (39, 146)]. A loss-of-function mutation in addition to sarcomere organization, recent studies indicate
human ankyrin B as well as heterozygous ankyrin B that nonsarcomeric actin is involved in vesicle trafficking
knockout mouse are associated with cardiac features in- (197), and a non-muscle tropomyosin (Tm5NM1) localized
cluding sinus bradycardia, abnormal heart rate variabil- adjacent to Z-line can define actin filaments that are asso-
ity, defects in cardiac conduction, and increased ventric- ciated with t-tubules, contributing to t-tubule organization
ular arrhythmia (148). The ankyrin B syndromes are dis- and function (219).
tinct from classical long QT syndrome, but can manifest
as sick sinus syndrome with bradycardia and increased Furthermore, BIN1⫹13⫹17 facilitates N-WASP-depen-
risk of sudden cardiac death. Results from cell biological dent actin polymerization, responsible for the maintenance
studies indicate that lack of ankyrin B microdomains of BIN1-microfolds at t-tubules. N-WASP is a ubiquitously
alters membrane ion channel localization to their physi- expressed protein involved in transduction of signals from
ological subdomains, disrupting intracellular calcium receptors to actin cytoskeleton. N-WASP is bound to the
homeostasis and promoting arrhythmias (24, 146, 148, actin nucleator Arp2/3 protein complex, and remains inac-
149). tive through autoinhibition. Upon activation by small
GTPase Cdc42, PIP2, amphiphysin 1, or BIN1, the func-
The composition, organization, function, regulation, and tional VCA domain of N-WASP is released from autoinhi-
turnover of these t-tubule-related membrane microdomains bition, which subsequently activates Arp2/3-dependent ac-
remain active areas of research. Further understanding of tin polymerization (203). In cardiomyocytes, N-WASP is
microdomain content and regulation may provide new in- localized at the z-discs (211). When interacting with t-tu-
sight into the biological functions of t-tubules in striated bule membrane associated BIN1⫹13⫹17, N-WASP activa-
muscles, both in normal physiology and during disease de- tion promotes F-actin polymer formation, stabilizing BIN1⫹
velopment. 13⫹17-membrane microfolds to z-discs (91).

2. Costamere complex and t-tubule membrane- diac BIN1⫹13⫹17 for the stabilization of t-tubule mi-
associated scaffolds crodomains around myofilament Z-discs. Whether sarco-
mere ␣-actin, costameric ␥-actin, and/or cortical ␤-actin
Costameres are cytoskeleton-rich structures surrounding fibers are involved in ␣-actinin-dependent microfold stabi-
the sarcomere Z-discs right below t-tubule membrane, serv- lization to Z-discs remains unidentified. Tcap is another
ing as anchor points between sarcolemma and myofilament. myofilament aligning protein (86, 111) that is critical in the
Costameres consist of two major protein complexes, the maintenance of the integrity of t-tubule network. Muta-
dystrophin-glycoprotein (DAG) complex and the vinculin- tions of Tcap have been associated with abnormal t-tubule
talin-integrin complex. The vinculin-talin-integrin complex morphology (111) and development of hypertrophic and
is involved in the bidirectional transmission of contractile dilated cardiomyopathies.
force between cardiomyocytes and extracellular matrix,
with vinculin as the protein linking membrane and actin Supported by these numerous membrane-associated pro-
cytoskeleton. By clustering with vinculin complex (106), teins, scaffolding proteins, cytoskeleton, costamere linking
dystrophin also has a primary mechanical role in mainte- proteins, sarcomere structural proteins, and extracellular
nance of cell membrane integrity. Dystrophin is a large matrix, t-tubules are well-organized rigid membrane organ-
427-kDa protein product encoded by the Duchenne muscu- elle with limited fluidity. Yet, t-tubule membrane system
lar dystrophy gene. In addition to the full-length dystrophin can also be extremely dynamic and labile (see more detailed
Dp427, a short isoform Dp71 is also expressed in mouse discussion in sect. V). It remains to be understood how these
cardiomyocytes (141). Dystrophin is present at cardiac t-tu- regulatory proteins work with each other to maintain a
bules from the time tubules develop. Dp427 is found in both rigid yet dynamic membrane system.
general sarcolemma and t-tubules, whereas Dp71 is specif-
ically expressed in t-tubules (58). The dystrophin-DAG
complex provides a structural link between cytoskeleton E. Variation in Cardiac T-Tubule Geometry
and extracellular matrix. Sarcolemma fragility secondary to
dystrophin degradation has been observed in dilated car- A high degree of variability in t-tubule geometry has been
diomyopathy (107). reported in myocytes from different cardiac chambers and
across species. T-tubules have been found in cardiomyo-
As discussed in other sections, t-tubule membrane-associ- cytes from all mammalian species studied so for (mouse, rat,
ated scaffolds such as BIN1 (sect. IIC), caveolin-3 (sect. rabbit, dog, sheep, pig, human), but are inconsistently re-
IIC), and junctophilin (sect. VA) are important to maintain ported or devoid from other phyla including amphibians,
normal cardiac t-tubule structure and function. Addition- reptiles, and birds (see review in Ref. 19). Across mamma-
ally, another membrane-inserted protein, dysferlin, is also lian species, it is well accepted that ventricular cardiomyo-
understood to be an important regulator of t-tubule mem- cytes have the most developed and mature t-tubule system
brane trafficking and repair during injury (6, 7, 46, 54). (see reviews in Refs. 19, 20, 80), whereas atrial myocytes
Dysferlin is a 230-kDa membrane inserted protein with have more scarce and irregular t-tubules (109, 121, 175,
binding affinity to both calcium and phospholipid (44), 215). Occasionally, t-tubules are identified to be present in
which is enriched in subsarcolemma vesicles and can be the Purkinje conducting system (51, 160). In mammalian
quickly recruited to the membrane injury site (7) to facili- ventricular cardiomyocytes, although the Z-line localiza-
tate membrane repair (54). In cardiomyocytes, dysferlin is tion of transverse tubules remains conserved across species,
found to be present in both sarcolemma t-tubules and inter- large variability is found in the geometry and size of t-tu-
calated discs (30). For t-tubule membrane repair, it has been bules. It appears that t-tubules in rodents and small mam-
proposed that upon insult, dysferlin can be recruited to mals are denser, deeper, and narrower with more structural
t-tubules along with its interacting partners including BIN1, complexity than t-tubules from large mammals (i.e., rabbit,
caveolin-3, and EHDs to induce repair (46). Without dys- pig, and human). Quantitative measurements of overall t-
ferlin, abnormal lipid vesicle trafficking impairs membrane tubule morphology and spacing, based on traditional fluo-
repair and accumulates neutral lipids like glycerol, which rescent microscopy which cannot discern membrane micro-
further promotes detubulation and myopathy (46). Dysfer- folds but is excellent and quantifying the larger structures,
lin deficiency is associated with dilated cardiomyopathy estimate segment branch length (L) to be ⬃9 ␮m and diam-
(84, 114, 226), further indicating its important role in main- eter (D) to be ⬃200 nm in mouse (91), ⬃7 ␮m (L) and
tenance of normal cardiac t-tubule function. ⬃250 nm (D) in rat (198), and ⬃2 ␮m (L) and ⬃400 nm
(D) in rabbit (183) and human (26) myocytes. The size and
3. Myofilament structural proteins complexity of t-tubule network seem to correlate with the
heart rate of each species (20). Conceptually, higher heart
The Z-disc organizing protein ␣-actinin, with the cardiac rates as in rodents require faster action potential propaga-
isoform being ␣-actinin 2, is the structural protein at both tion into cell interior, allowing for efficient intracellular
Z-disks and peri-Z-disc costameres. Using F-actin filament, Ca2⫹ diffusion to SR to maintain the synchronicity and
␣-actinin is linked to the t-tubule membrane-associated car- efficacy of beat-to-beat myofilament contraction (16, 20,

237). Humans and large mammals have resting heart rates diomyocytes identified the relative amount of membrane
less than 100 beats/min, so relatively larger and more currents at t-tubule membrane versus non-t-tubule sarco-
widely spaced t-tubules (20, 90) can fulfill the same func- lemma (see review in Ref. 19).
tional needs to maintain synchronized ventricular contrac-
tion during each heartbeat. 1. Ca2⫹ handling proteins
Although a t-tubule network is a membrane structure com-

The most cardiac t-tubule “signature” protein is the cal-
mon to the striated muscles, cardiac t-tubules have unique
cium transient initiator LTCC, consisting of one ␣ subunit
characteristics that are distinct with regard to tubular size,
with multiple auxiliary ␤, ␣2␦1, and ␥ subunits. The ICa
location, and numerical count. Compared with the small
conducting pore is formed by the large 24 transmembrane
skeletal tubules with diameter averaged at 20 – 40 nm, car-
domain containing an ␣-subunit, whose trafficking and gat-
diac t-tubules are much larger with estimated diameter to be
ing properties are regulated by the auxiliary subunits (42).
100 – 400 nm depending on species. Meanwhile, cardiac
t-tubules are present within 0.5 ␮m of Z-discs (198), The predominant splice variant of ␣-subunit expressed in
whereas skeletal t-tubules are localized to the interface be- cardiomyocytes is ␣1c, or CaV1.2 (212). CaV1.2 LTCCs are
tween A band and I band of the sarcomere. Thus cardiac critical in cardiac development and function (186). Global
t-tubules appear once every sarcomere length spaced out at deletion of CaV1.2 induces abnormal cardiac morphogene-
⬃1.8 –2 ␮m along the longitudinal edge of the myocytes. sis with embryonic lethality (186). On the other hand, in
The skeletal t-tubules, on the other hand, consist of two sets postdevelopment adult hearts, inducible deletion of CaV1.2
of transverse tubules at each A band/I band interface within leads to reduced contractility and increased susceptibility to
individual sarcomeres of skeletal myofilaments. stress-induced heart failure (76). As the cell surface dyadic
channel, LTCCs need to be concentrated to t-tubules for
better coupling with RyRs from jSR membrane, allowing
III. TRANSMEMBRANE PROTEINS AT efficient CICR and normal EC coupling (163). Immunocy-
CARDIAC T-TUBULES tochemistry data indicate that LTCCs are concentrated at
t-tubules, and electrophysiology measurement identified
Continuously extended from surface sarcolemma, t-tubules that 80% of the surface calcium current mediated by LTCC
are lipid bilayers embedded with transmembrane or lipid- is at t-tubules. Such a high concentration of LTCCs at t-tu-
associated proteins. Studies indicate t-tubules have a differ- bule membrane is achieved by microtubule-dependent tar-
ent lipid composition relative to general sarcolemma. Pre- geted delivery of LTCCs to t-tubules, a process facilitated
vious studies from skeletal muscle identified that t-tubules by the t-tubule membrane curvature protein BIN1 (94) (FIG-
are enriched in cholesterol (27) and phospholipids, and URE 4). Furthermore, LTCCs at t-tubules are not distrib-
have a higher phospholipids/cholesterol ratio (167, 178, uted evenly, but rather clustered to membrane microdo-
204), with phosphotidylserine and sphingomyelin as the mains formed by BIN1-microfolds. These LTCCs clusters
two most abundant phospholipid species. The enrichment will couple with RyRs channels on the nearby jSR mem-
of phospholipids may be enhanced by the BIN1 organized brane to form functional calcium releasing units bridged by
membrane microfolds which preferentially bind to and con- BIN1 molecules (FIGURE 4). These TEM visible electron-
centrate negatively charged phosphoinositides (PIP2) (120). dense feetlike structures, dyads, are the initiation sites
The full lipid profile of cardiac t-tubules awaits careful lip- where beat-to-beat calcium transient starts. During each
idomic studies. In contrast, the protein components of t-tu- action potential, membrane depolarization activates volt-
bules are well studied. This section will focus on the current age-gated LTCCs to induce calcium influx. The initial cal-
knowledge of the transmembrane protein composition at cium ions entered through LTCCs at the junctional dyads
t-tubule membrane, focusing on ion handling proteins and diffuse a short distance to activate the nearby calcium sens-
signaling molecules. ing and releasing RyRs at jSR membrane, inducing a large
release of calcium from SR lumen to cytosol. This is a se-
quence of events known as calcium-induced calcium release
A. Ion Handling Proteins (CICR). How LTCC-RyR dyads are organized by BIN1-
(Channels/Transporters/Pumps) microfolds to control CICR and EC coupling (66) will be
further discussed in section IV focusing on the functional
The expression of transmembrane ion channels, ion trans- regulation of dyads.
porters, and pumps have been well characterized in cardiac
t-tubules. The calcium handling proteins that are important During relaxation, distal SR membrane localized sarco/en-
in cardiac EC coupling, in particular the LTCCs, are mostly doplasmic reticulum Ca2⫹-ATPase (SERCA) reuptake ele-
enriched in t-tubules. Other Na⫹ and K⫹ channels and han- vated cytosol calcium into SR storage. Meanwhile, the ele-
dling proteins are also found to be present in t-tubules, vated intracellular calcium at the cleft between t-tubule and
although to a lesser degree of enrichment. Electrophysiol- SR membrane will also be extruded through sarcolemmal
ogy studies in normal and osmotic shock detubulated car- NCX1, a calcium handling protein anchored at ankyrin-B

microdomains within t-tubules that is critical for intracel- ankyrin B. Distinct from BIN1-organzied LTCC-RyR dy-
lular calcium homeostasis. ads, ankryin B similarly tethers cytoskeleton to facilitate
targeted delivery of NCX1 and NKA (147), resulting in the
2. Na⫹ handling proteins concentrated expression of the functional NKA and NCX1
at t-tubules (⬃3- to 3.5-fold higher than external sarco-
In most excitable cells, the voltage-gated sodium channel- lemma) (48).
mediated Na⫹ current is responsible for the rapid action
potential upstroke. Sodium channels consist of four pore- 3. K⫹ handling proteins
forming ␣-subunits and one or two auxiliary ␤-subunits.
Ventricular cardiomyocytes express several different pore- Most potassium channels are more uniformly distributed
forming subunits of the voltage-gated Na⫹ channels includ- between t-tubules and surface sarcolemma in cardiomyo-
ing the primary cardiac isoform Nav1.5 and the brain-type cytes. Detubulation of cardiomyocytes only decreases the
isoforms Nav1.1, Nav1.3, and Nav1.6 (136), with different steady-state current Iss [mediated by Kv1.2/2.1 (184) or
intracellular localization and functional importance. So- TASK-1 (104)], without affecting the density of other po-
dium current measured in control and detubulated rat ven- tassium currents including transient outward current Ito
tricular cardiomyocytes indicate more uniform distribution (mediated by Kv4.2/Kv4.3), inward rectifier current
of cardiac Nav1.5 current along sarcolemma and t-tubules Ik1 (mediated by Kir2.x), and delayed rectifier current Ik (Ikr
(in channels/␮m2, 11 on total sarcolemma vs. 10 on t-tu- mediated by HERG, and Iks mediated by KvLQT1) (113).
bules), and a significant t-tubule concentration of current Interestingly, immunofluorescence and TEM imaging data
mediated by neuronal sodium channel isoforms (in chan- demonstrated that the Ito generating Kv4.2 is concentrated
nels/␮m2, 1 on total sarcolemma vs. 2.5 on t-tubules). at both t-tubules and intercalated discs (8). The lack of
current density change after detubulation may be due to the
Although Nav1.5 is also localized to cardiac t-tubules (55), Ito current at the intercalated discs. Antibody labeling also
this primary cardiac ␣-subunit Nav1.5 is preferentially lo- supports that Ik1 generating Kir2.x, including Kir2.1 (35),
calized to intercalated discs with auxiliary ␤2- and/or ␤4- Kir2.2 (122), and Kir2.3 (143) channels, are localized to
subunits, mediating rapid action potential propagation t-tubules. Other ATP-sensitive inward rectifiers (Kir6.x) are
from cell to cell across the myocardium. In contrast, the also localized to t-tubules. Both IKATP and Ik1 decrease
brain isoforms of Nav1.1, Nav1.3, and Nav1.6 together along with t-tubule membrane loss when cardiomyocytes
with ␤1- and/or ␤3-subunits, are localized to t-tubules (135) are cultured in vitro (34, 145). Interestingly, KATP channel
and near t-tubule openings (56). These t-tubule localized release from the Golgi can be dependent on stress-induced
brain Nav isoforms have more negative voltage dependence PKA activation, resulting in rapid insertion of KATP chan-
of gating and more rapid activation and inactivation kinet- nels into t-tubule membrane (1). If the cargo is in the close
ics, facilitating fast action potential inward penetration proximity to the membrane, there may a direct vesicle-me-
from myocyte surface to interior via t-tubule invagination. diated Golgi to membrane translocation pathway (172).
However, the role of these channels in EC coupling remains Immunofluorescence data also indicate that the cardiac Ikr
controversial. T-tubule localized neuronal isoforms of so- channels, which consist of two ␣-subunits hERG1a and
dium channels have been indicated as contributors to more hERG1b, are localized to t-tubules (103).
effective CICR in some studies (136), and removal of
Nav1.6 has been shown to prolong calcium transient dura-
tion (155). Other studies indicate that, although these neu- B. Signaling Molecules at Cardiac T-Tubules
ronal sodium channels are concentrated at cardiac t-tu-
bules, they are not required for EC coupling (21). The most well-studied signaling pathway at t-tubules is the
G protein-coupled ␤-adrenergic receptor (␤-AR) pathway,
NKA uses ATP hydrolysis-derived energy to pump 3 Na⫹ which mediates the sympathetic control of myocardial
and 2 K⫹ ions across membrane, hence maintaining mem- function. Cardiomyocytes express both ␤1-AR and ␤2-AR,
brane potential in cardiomyocytes. Three catalytic ␣ (␣1, which are coupled to the Gs-AC-cAMP cascade (see review
␣2, ␣3) subunits of NKA are expressed in the heart, with ␣1 in Ref. 233). Through the activation of cAMP-dependent
as the dominant isoform (206). However, the ␣1 isoform is protein kinase A (PKA) and the subsequent phosphoryla-
more uniformly expressed, whereas isoforms ␣2 and ␣3 are tion by PKA of critical proteins involved in calcium han-
mainly located in the junctional membrane within t-tubules dling and contractile function, ␤-ARs modulate catechol-
(105). Studies have shown that isoform ␣2 expression is amine-dependent rate, force, and speed of myocyte contrac-
four times higher in t-tubules versus non-t-tubule sarco- tion. The protein targets of ␤-AR-activated PKA include
lemma (10). Within t-tubules, NKA colocalizes with NCX1 sarcolemmal t-tubule LTCCs (67, 68), SR membrane RyRs
at ankyrin B microdomains, contributing to the regulation (139) and phospholamban (PLB) (125), and intracellular
of CICR and calcium transient. NKA and NCX1 and the myofilament proteins (troponin I and C proteins) (12). In
jSR localized InsP3 receptors are clustered to a junctional addition to the stimulatory Gs pathway which ␤1-AR is
subdomain organized by a membrane scaffolding protein solely coupled to, ␤2-AR is also known to couple to the

inhibitory Gi protein (234, 235). Thus, within ␤2-AR-en- research since late 1980s. The dyad membrane structure
riched caveolae (14, 229) and t-tubule (77), the ␤2-AR to Gi formed by t-tubules and jSR terminal cisternae is critical in
pathway can negate ␤2-AR-Gs mediated cAMP-PKA signal- organizing EC coupling of heart muscle. In 1989, Fabiato
ing. (59) proposed the model of CICR as the molecular mecha-
nism underlying EC coupling, which is further supported by
In the t-tubule system, several factors affect the compart- the later identification of calcium sparks as the elementary
mentalization of the ␤-AR signaling. First, the differential events (31). The sarcolemma voltage-gated LTCCs, which
distribution pattern of ␤1-AR and ␤2-AR reveals that are activated during membrane depolarization following
␤1-AR is homogeneously spread throughout sarcolemma each action potential, conduct the initial calcium influx to
and t-tubules, whereas ␤2-AR is more selectively confined induce the subsequent series of events of intracellular CICR.
to t-tubules (77, 154). Second, LTCCs, the ion channel The intracellular calcium sense and release units are RyRs
targets of Gs-activated PKA, are concentrated to t-tubule on the jSR membrane. In addition to dyad formation for
membrane with particular clustering to BIN1-microfolds efficient CICR and normal EC coupling, t-tubules also cre-
(94). Third, a number of signaling molecules in the ␤-AR- ate a diffusion barrier for extracellular ions, protecting elec-
Gs-PKA cascade are also localized to t-tubules, including trical stability of the cardiomyocytes during fluctuations in
phosphodiesterase 3 (PDE3), PKA, AKAPs, and AC. The extracellular bulk environment. The recently identified
␤1/2-ARs mediated Gs-cAMP signaling at t-tubules is there- BIN1 microdomains contribute to both dyad microdomain
fore more confined to subsarcolemmal microenvironment formation (65, 94) and generation of a “fuzzy space” (119)
right below the t-tubule openings, where most BIN1- restricted diffusion zone for extracellular ions, thus serving
microfold clustered dyadic LTCCs and RyRs are also as a major regulator of the primary functions of cardiac
enriched. The structural restriction of AKAP on PKA t-tubules (FIGURE 2).
diffusion (78, 110) further confines ␤-AR-Gs-PKA signals
close to submembrane microdomains to more effectively A. Dyad and EC Coupling
modulate the phosphorylation and activity of surface
LTCCs without affecting intracellular protein targets. Normal ventricular EC coupling requires a calcium tran-
Compartmentalization of ␤-AR signaling to cardiac dy- sient initiated at the cardiac t-tubules. As discussed earlier,
ads is also linked by cBIN1-microdomains, which both the current accepted model of intracellular calcium tran-
organize LTCC-RyR dyads and undergo fast reassembly sient development is CICR (59). At each heartbeat, in re-
upon acute ␤-adrenergic activation (66). sponse to action potential-triggered inward sodium current,
sarcolemmal membrane depolarization activates voltage-
Other than the secondary messenger cAMP-dependent gated LTCCs at the t-tubule membrane. The initial calcium
function in contractile regulation, ␤-AR signaling pathway influx mediated by activated LTCC subsequently induces a
also contributes to regulation in apoptosis and cell survival. massive calcium release from SR stores. Proposed as early as
The ␤1/2-AR-cAMP-PKA pathway stimulates apoptosis by in late 1980s, this CICR model was further supplemented
activating MAPK family, particularly ERK1 and ERK2 (32, by the identification of the jSR membrane localized RyRs
41, 241). The antiapoptotic effect of ␤2-AR activation, on (100, 168). In the early 1990s, calcium sparks from the SR
the other hand, is more likely mediated through the G␤␥ were then identified as the “elementary units” of the cal-
subunits, which are released from Gi upon ␤2-AR activation cium transient (31). The complexes consisting of LTCCs at
by catecholamine (240). Thus t-tubule localized ␤2-AR also t-tubules and RyRs at jSR membrane (⬃1:4 ratio) form
signal through Gi-G␤␥ pathway to activate the pro-survival cardiac dyads (13, 25). Close physical association of jSR
PI3K and AKT signals (240). membrane localized RyRs with sarcolemma LTCCs (31,
59, 100, 168) is required for optimal CICR needed for
Given the differential role of ␤1-AR/␤2-AR in regulating cell efficient EC coupling. Such a close association between the
contractile function and apoptosis/cell survival, t-tubule- two dyadic channels is achieved by LTCC localization to
dependent-compartmentalized ␤2-ARs play a critical role in t-tubules (11, 185) and clustering to microdomainds in-
regulating these signaling pathways to maintain normal duced by BIN1 microfolds, which also recruit RyRs to jSR
cardiac function. In fact, redistribution of ␤2-AR with al- membrane for LTCC-RyR couplon formation (FIGURE 3).
tered ␤2-AR-cAMP signaling occurs during heart failure Upon membrane depolarization, the initial calcium influx
(77, 134, 154), indicating the importance of t-tubule com- through LTCCs and the close association between LTCCs
partmentalized ␤2-AR-cAMP signaling in normal cardiac and RyRs (⬃15 nm) at dyads permit efficient CICR and
function and during disease development. subsequent sarcomeric contraction (156).
1. LTCC trafficking and clustering to dyads through

IV. FUNCTION OF CARDIAC T-TUBULES BIN1
Exploration of the functional importance of t-tubules in The calcium transient initiator LTCCs need to be concen-
cardiac calcium transients has been a very active field of trated and clustered to t-tubule microdomains for better

coupling with RyRs to form functional dyads (163). Precise (see sect. II). Compelling data are that deletion of the coiled-
t-tubule localization of LTCCs relies on the membrane scaf- coil and SH3 domains abrogates the ability of BIN1 in
folding protein BIN1 for several reasons. First of all, LTCC facilitating Cav1.2 trafficking without altering membrane
trafficking from the Golgi apparatus occurs on microtu- invagination (94). Thus targeted delivery of Cav1.2 is
bules which deliver LTCCs to BIN1 containing membrane achieved through interaction with the BIN1 molecules at
(94). This microtubule-dependent channel delivery high- t-tubules, rather than the t-tubule structures themselves.
way with specificity, known as targeted delivery, was intro- The non-BAR domains in BIN1 molecules likely attract
duced via previous studies using connexin 43 trafficking to Cav1.2 channels through interaction with the COOH ter-
intercalated discs as a model system (93, 94, 188, 195). minus of the channel. The involvement of BIN1 in traffick-
Briefly, targeted delivery describes that, after exiting the ing Cav1.2 is further confirmed in a series of subsequent
Golgi, membrane vesicles containing transmembrane ion studies using BIN1-deficient ventricular cardiomyocytes
channels are rapidly directed on microtubule highways (91, 93). Alternative translation of connexin 43 provides
across the cytoplasm to their specific membrane microdo- smaller isoforms (FIGURE 6) that assist with forward traf-
mains (FIGURE 5). The microtubules with negative ends are ficking of the full-length protein (196). Fragments of the
anchored at the microtubule organizing center (MTOC) LTCC COOH terminus occur and have been attributed to
near Golgi, and the fast growing positive ends are ap- cleavage (69). It is not known if these fragments could be
proaching outward ready to be captured by membrane an- formed by alternative translation instead, if whether they
chor protein complexes at specific plasma membrane sub- assist with forward trafficking.
domains. Specificity of delivery requires a combination of
the individual channel protein or truncated isoform (FIGURE In addition to facilitate forward trafficking of Cav1.2 chan-
6), the plus-end-tracking proteins at the growing ends of nels, BIN1 also plays a critical role in clustering Cav1.2
microtubules, and the membrane anchor protein complex channels that are already localized at the t-tubule mem-
responsible for capturing. In the case of Cav1.2 channel brane. Through formation of BIN1 microfolds, these
delivery, t-tubule-associated BIN1 anchors growing micro- unique membrane structures form curved membrane mi-
tubules with Cav1.2 cargoes, allowing the delivery of crodomains enriched with Cav1.2 channels. As indicated in
Cav1.2 to occur at BIN1 molecules (94). BIN1 contains a our recent super-resolution STORM imaging (XY-resolu-
membrane curvature BAR domain, the middle coiled-coil tion at 10 –20 nm, Z-resolution at 50 nm), Cav1.2 form
regions, and an SH3 protein-protein interaction domain discrete clusters at cuplike microdomains created by BIN1
Extracellular space T-tubules
Cytoplasm Cell 1 Cell 2
Intercalated disc
FIGURE 5. Schematic representation of
ion channel targeted delivery. Channel pro-
BIN1 teins are sorted into vesicular carriers and
docked onto microtubules at the trans-
Golgi network (TGN) and subsequently de-
livered to their subcellular destinations in
cooperation with actin “rest stops” along
Actin Cx43 the route. Microtubule plus-end binding
proteins interact with anchor proteins of
specific membrane subdomains, allowing
targeted delivery of cargo proteins. In the
case of connexin 43 (Cx43) trafficking, the
interaction between the microtubule plus-
end binding protein EB1, the channel itself,
and the adherens junction complex en-
LTCC
sures the targeted delivery of Cx43 hemi-
channels to the intercalated discs. For
LTCC delivery to t-tubules, key components
are the LTCC channels, a ⫹TIP protein, and
Microtubule cardiac bridging integrator 1 (cBIN1). The
+Tip protein
microtubule ⫹TIP protein associated with
LTCC delivery has not yet been identified.
Golgi

GJA1-43k GJA1-32k GJA1-29k GJA1-26k GJA1-20k GJA1-11k GJA1-7k
M213
M100 M147
M125 M281
NH2 M320
M1
HOOC HOOC HOOC HOOC HOOC HOOC HOOC
Transmembrane domain Methionine location corresponding to the AUG start site
FIGURE 6. Alternative translation of connexin 43. In addition to full-length 43-kDa connexin 43 (GJA1-43K),
there are six NH2-terminal truncated smaller Cx43 isoforms (GJA1-32K, GJA1-29K, GJA1-26K, GJA1-20K,
GJA1-11K, and GJA1-7K) resulting from internal methionine residues that initiate ribosomal translation, a
process known as alternative translation. At least one of the isoform, GJA1-20k, has been identified as
necessary for the full-length channel to traffick to the surface membrane.
microfolds. The role of BIN1 in clustering Cav1.2 channels concentration, receptor phosphorylation, as well as reorga-
is further supported by the observed smaller Cav1.2 clusters nization of BIN1 microdomains (2). It was recently identi-
in adult mouse ventricular cardiomyocytes with Bin1 fied that t-tubule-attached BIN1 recruits RyRs into dy-
heterozygous deletion (91). Clustering of Cav1.2 channels ads, an interaction that is enhanced after RyR phosphor-
at these t-tubule microfolds alters channel kinetic properties ylation (66). The enrichment of positively charged
including gating and open probability (53, 152). How BIN1 residues within the BAR domain of BIN1 may explain its
microdomains regulated Cav1.2 clustering alters coupled preferential binding to negatively charged hyperphos-
gating of LTCCs at the t-tubule surface is an interesting and phorylated receptors, in a way similar to the known bind-
important area of future consideration. ing affinity of BIN1 to negatively charged phospholipids.
Interestingly, upon acute ␤-AR activation, BIN1 is redis-
2. RyR recruitment to dyads through BIN1 tributed to t-tubules, a result likely due to ␤-AR activa-
tion-induced phospholipid accumulation at the t-tubule
The other critical dyadic component is the RyR homote- lipid bilayer. Subsequently, phosphorylated RyRs are
tramer at the jSR membrane apposing LTCCs at t-tubules. further recruited to BIN1 microdomains, allowing effi-
In the RyR tetramer, the COOH-terminal transmembrane cient RyR coupling with LTCC for functional dyad for-
pore domain of each RyR monomer comes together to form mation, improving EC coupling with preserved electrical
a calcium-conducting pore across the SR membrane. The stability during acute stress (FIGURE 7) (66).
four NH2-terminal cytoplasmic domains from the tetramer
constitute a large mushroomlike platform containing bind- 3. Junctophilin-2 in t-tubule-jSR junctional membrane
ing sites (182) for various modulators including calstabin complex formation
(216), PKA and mAKAP (muscle A kinase anchoring pro-
tein), phosphatases PP1 and PP2A (139, 140), as well as Another well-studied protein family involved in t-tubule
calmodulin (236) and Ca2⫹/calmodulin kinase II (CaMKII) and jSR organization is the junctophilins (JPs), which have
(223). Biophysical properties of RyR can be regulated by the apparently unique ability to directly bind to both t-tu-
these modulators via alteration in receptor posttransla- bule and SR membranes. In mammalian tissue, three junc-
tional modifications including phosphorylation states, lu- tophilin proteins JP-1, JP-2, and JP-3 are encoded by differ-
minal calcium (81), or stabilization of receptor closed ent genes, with JP-2 expressed in heart. JP-2 has a COOH-
states. The macromolecular complex centering on RyR terminal hydrophobic segment spanning the SR membrane,
channels facilitates efficient spatial and timely regulation of a linkage ␣-helical domain, and the remaining cytoplasmic
channel function. region consisting of eight lipophilic “membrane occupation
and recognition nexus (MORN)” domains (115). The
It is important to note that the arrangement of RyR at dyad MORN domains are associated with t-tubules, approxi-
is neither uniform nor static. The receptor cluster contains mating t-tubule sarcolemma to the jSR membrane. It re-
10 –300 RyR homotetromers (63) with a spatial separation mains unclear whether JP-2 facilitates BIN1 bridged LTCC-
of 0.6 –1 ␮m between clusters (199), the organization of RyR dyad formation and function. Whether JP-2 is critical
which can be altered on a time scale of minutes by Mg2⫹ in EC coupling and contributes to heart failure progression

Baseline Isoproterenol
TT
SR lumen
Wildtype
BIN1
Cav1.2
Bin1 HT
dP-RyR P-RyR Orphaned P-RyR
FIGURE 7. Cartoon of BIN1-dependent recruitment of phosphorylated ryanodine receptors (RyR) to dyads

during acute stress. Acute isoproterenol (ISO) redistributes BIN1 to cardiac t-tubules and subsequently
recruits phosphorylated RyRs to couple with LTCCs at dyads, increasing excitation-contraction coupling gain
while maintaining electrical stability. In Bin1 HT cardiomyocytes with less BIN1 available, insufficient RyR
recruitment leads to accumulation of hyperactive phosphorylated RyRs outside of dyads, increasing sponta-
neous calcium release and promoting arrhythmias.
is also controversial. Some studies indicate that JP-2 is evidence indicates that extracellular ions diffuse slowly
downregulated in animal models of hypertrophic and di- within the t-tubule network, compared with extracellular
lated cardiomyopathy, and conditional knockdown of JP-2 bulk environment. In fact, ion diffusion coefficients within
in the heart causes systolic heart failure in murine models the t-tubule network are normally 5–10 times slower than
(218). Other studies report no significant changes of JP-2 those in the extracellular bulk environment (189, 209).
expression in failing hearts (23), or with recovery of heart Fluctuations in environmental extracellular ion concentra-
function (134). tions therefore occur with delayed lag phase before reaching
to t-tubule membrane and its complement of ion channels.
B. Ion Diffusion and Membrane Excitability For instance, the change of extracellular calcium is delayed
by up to 2.3 s to reach the t-tubules in guinea pig myocytes
One of the functional attributes of t-tubules is the presence (16). This lag phase due to restricted diffusion within the
of slow diffusion zones close to t-tubule sarcolemma (164, t-tubules can have a significant effect on muscle cell homeo-
189, 209). Restricted diffusion occurs both at extracellular stasis and stability, particularly during environmental fluc-
and intracellular spaces near the t-tubule membrane with tuations induced by acute stress. Increased heart rate and
accumulated ions such as calcium, which was originally quick transmembrane ion flux together with limited diffu-
observed as a theoretical “fuzzy space” (119). Growing sion can rapidly accumulate outward current ions like po-

tassium (209) and deplete inward current ions like calcium for achieving timely intracellular calcium equilibrium re-
(164, 165), affecting the driving force of ion channels at quired for beat-to-beat heart contraction. Recent evidence
t-tubule membrane and shortening the action potential du- indicates that crosstalk between BIN1 microdomains and
ration (166). Shortened action potential limits t-tubule caveolae likely exists for better control of ␤-adrenergic-
membrane excitability and electrical instability, serving as a regulated calcium entry through LTCCs, especially during
crucial protective mechanism in preventing detrimental acute stress as well as chronic dysregulation in heart failure.
ventricular arrhythmias. It is likely that caveloae microdomains are indeed related to
BIN1 microfolds, since intracellular localization of caveo-
Although t-tubule forms a complex interconnected network lin-3 is altered in cardiomyocytes missing the Bin1 allele
(198), this network feature does not explain the highly re- (116). The role of BIN1 in regulation of caveolae localiza-
stricted diffusion near the t-tubule sarcolemma. The struction and function remains to be tested. Nevertheless, in
tural foundation of the restricted diffusion zone was not addition to organizing microfolds-based dyads, BIN1 may
revealed until the recent discovery of t-tubule membrane have a significant role in other t-tubule microdomains as
microfolds created by cardiac BIN1⫹13⫹17. In wild-type well.
adult mouse ventricular cardiomyocytes, along with tortu-
ous t-tubule (91) path meandering into the myocyte inte-
rior, BIN1 microfolds are present at t-tubule membrane, V. LIFE CYCLE OF CARDIAC T-TUBULES
bringing spatial complexity to the lumen. These spatial di-
vided subdomains with narrow connecting points to the Despite their structural complexity, t-tubules are extremely
central lumen help trap extracellular ions, preventing ionic labile (112). T-tubules are absent in embryonic myocytes
diffusion. For example, the small extracellular gaps be- (187); develop only after birth (82, 187); dedifferentiate in
tween layered membrane microfolds can trap Ca2⫹ and K⫹. cultured myocytes (118, 130, 145), a process that can be
Thus, in addition to dyad microdomain organization, BIN1 slowed by actin stabilization (118, 214); remodel during
microfolds also create spatial pockets, serving as reservoirs heart hypertrophy and failing (130, 132, 133, 224); and can
for extracellular ions and other signaling molecules. In recover during functional recovery of the hearts (133). In
BIN1-deficient cardiomyocytes, these microfolds and so addition to membrane turnover, other t-tubule components
formed spatial pockets are lost and tortuous t-tubules be- including transmembrane ion channels and receptors are
come more straight and dilated, normalizing rapid ion dif- also extremely dynamic. By regulating protein trafficking
fusion at t-tubules. Mathematical modeling further con- and recycling, cardiomyocytes maintain a steady state of
firms that removal of this diffusion barrier increases fast ion continuously replenished t-tubule pool of ion channels and
diffusion from t-tubule network to outside bulk environ- signaling molecules for functional homeostasis. Mean-
ment. When BIN1 is reduced as occurs in heart failure, loss while, intracellular cytoskeleton components including cor-
of BIN1 microfolds removes the protective ion diffusion tical actin, microtubules, and costameres are actively regu-
barrier, increasing membrane excitability and promoting lated to control membrane integrity and protein function.
arrhythmias (91). In short, the microfold-forming cardiac This flexible function of t-tubules allows them to accommo-
BIN1⫹13⫹17 plays a pivotal role in maintaining and or- date both healthy and stress environments.
ganizing t-tubule ultrastructure essential for cardiomyocyte
electrical stability and homeostasis. Interestingly, recent
studies in zebrafish skeletal muscle indicate that skeletal A. Membrane Biogenesis, Maintenance, and
BIN1 may also have a similar function in maintaining t-tu- Turnover
bule ultrastructure (194). Whether a similar membrane mi-
crodomain-formed slow diffusion zone exists in narrow Unlike the sarcoplasmic reticulum membrane system,
skeletal tubular network with functional significance re- which develops in the fetal stage, cardiac t-tubules are ab-
mains to be identified. sent in embryonic hearts. In rodent and rabbit hearts, the
process of t-tubule biogenesis initiates a few days after birth
(82, 83, 187), along with the rise of the left ventricular
C. Other Microdomain-Related Functions pressure and working volume. Mature t-tubules are only
fully developed by the end of the first month of life. The
Other t-tubule microdomains, in addition to dyads, serve as mechanism of cardiac t-tubule development remains un-
signaling centers by concentrating transmembrane recep- clear. The Franzini-Armstrong group proposed in 2007 that
tors, ion channels, and signaling molecules. For instance, the inward penetration of t-tubule invagination is driven by
caveolae are enriched with a subset of LTCCs (4) as well as accrual of membrane lipids and specific proteins (50). Up to
␤2-AR (229), permitting efficient calcium signaling regula- this date, limited progress has been made to further under-
tion by the ␤-adrenergic system (5). In addition, ankyrin stand the lipids, proteins, and other molecules involved in
B-organized NCX/NKA/InsP3 macromolecular complex biogenesis process of t-tubules. The need to resolve the mo-
(147) facilitates calcium removal during muscle relaxation. lecular mechanisms of postnatal cardiac t-tubule develop-
Coordination of these t-tubule microdomains is necessary ment becomes even more urgent since the fast development

in the stem cell research field over the last few years. In calcium regulatory microdomains at t-tubule membrane.
2007, Yamanaka’s group successfully reprogrammed dif- The equilibrium between microfold organization and turn-
ferentiated human somatic cells into an undifferentiated over is achieved by the amount of BIN1 protein at the
pluripotent state for the generation of induced pluripotent t-tubule membrane. The released BIN1-containing micro-
stem (iPS) cells (210), which can then be redifferentiated particles therefore carry a signature of cardiomyocyte t-tu-
into most any terminally differentiated cell types including bule membrane, which can be quantified in plasma level,
cardiomyocytes. These iPS-induced cardiomyocytes con- and the resulted plasma cardiac BIN1 level can be used as a
tain essential contractile apparatus including sarcomeres, marker of myocardial health (92). How this BIN1-mediated
yet they do not have a mature t-tubule network. It is of great membrane turnover and release is linked to other mem-
interest to know how to mature these iPS-differentiated brane vesicle trafficking processes, such as injury-related
cardiomyocytes into adult phenotype with characteristic in- membrane repair (45, 46) especially during acute stress and
terconnected t-tubules and efficient EC coupling machiner- insult, will be interesting to pursue in the future.
ies.
T-tubule networks and especially BIN1 organized microdo- B. Ion Channel Trafficking and Half-Life
mains can also be extremely dynamic. The gross t-tubule
network in isolated cardiomyocytes loses integrity when Transmembrane ion channels and ion handling proteins are
cardiac origin cells are cultured in vitro, and t-tubule loss dynamically turned over to maintain a functional pool at
and remodeling are also a signature of failing cardiomyo- the t-tubule surface. In cardiomyocytes, the half-life of in-
cytes. On one hand, the SH3 domain of BIN1 binds to dividual ion channels is remarkably short, on the order of
dynamin 2 and is involved in dynamin 2-dependent endo- hours. The half-lives of t-tubule localized calcium and po-
cytosis (22, 171), indicating a possible intracellular recy- tassium channels, and sodium-calcium exchangers, are all
cling of BIN1 microfolds through endocytic vesicle forma- on the order of 10 h (33, 37, 49, 57). Even the more ex-
tion (FIGURE 8, adapted from BAR-regulated endocytosis in tended half-life of sodium channels is ⬃35 h (138). For an
non-cardiac cells in Ref. 232). On the other hand, the re- overall turnover scale of several to tens of hours, the bulk of
cently identified BIN1 microfolds are lost in isolated cardi- the ion channels in each cardiomyocyte are regenerated and
omyocytes during extended in vitro culture (91), indicating have to be newly positioned to t-tubule membrane in the
external release of BIN1 microfolds. Loss of BIN1 micro- course of any day. To maintain such a steady state and
folds can be rescued by replenishment of the microfold- continuously replenished t-tubule pool for functional ho-
forming BIN1 isoform and stabilization of the filamentous meostasis, ion channels at the t-tubule membrane need to be
actin cytoskeleton. Furthermore, BIN1 is blood available, tightly regulated at both forward trafficking and recycling
and plasma BIN1 correlates with cardiac function (92). It is pathways. In fact, ion channels often contain multiple sig-
possible that plasma BIN1 originates from cardiac t-tubules naling sequences that regulate forward trafficking including
as a result of BIN1-microfold turnover and release. In ad- the rate of their synthesis, organization at endoplasmic re-
dition to stabilizing membrane microfolds to Z-discs, BIN1 ticulum such as multimerization with auxiliary subunits,
may also regulate cytoskeleton dynamics for microparticle and traffic through Golgi apparatus before arrival at the
release (see speculative release mechanism in FIGURE 9), plasma membrane (144, 205, 239). On the other hand,
balancing the amount of microfolds and microfold-based t-tubule pool of ion channels is also regulated by retrograde
Extracellular space
T-tubule
Actin
Dynamin 2
BIN1
Cytoplasm
FIGURE 8. Schematic of endocytosis from cBIN1 microdomains. Adapting BAR domain regulated endocy-
tosis from noncardiac cells (229), we expect that for t-tubules, cBIN1 interacts with cortical actin to generate
an endocytic neck, and the SH3 domain of BIN1 the binds to dynamin 2 for scission and internalization of the
endocytic vesicle.

Extracellular space
T-tubule
Actin
BIN1-MP
BIN1
Cytoplasm
FIGURE 9. BIN1-microdomain release from cardiac t-tubules. A schematic of how fission can potentially
occur between two neighboring invaginated cBIN1 microfolds, resulting in external release of cBIN1 microfolds
that result in blood-borne microparticles.
trafficking including internalization, recycling, and degra- lion patients worldwide (129, 176, 177), particularly in the
dation. Furthermore, it is possible that ion channel half-life developed countries including the United States. The pri-
at plasma membrane may occur at a different time scale. mary mortality involves cardiac pump failure as a conse-
For instance, the half-life of active NCX in the plasma mem- quence of reduced cardiac contractility and sudden cardiac
brane is likely to be under 12 h (57, 127), which is shorter death due to detrimental ventricular arrhythmias. The de-
than the long half-life of 33 h measured using metabolic finitive therapy for end-stage heart failure is a heart trans-
labeling in lysates from neonatal cardiomyocytes (193). plant, which unfortunately remains as a limited option due
Similarly, the functional half-life of the Cav1.2 channel is to poor organ availability (102), and a need for subsequent
⬃3 h (33), whereas the total protein turnover occurs at a care and monitoring. The alternative therapy for end-stage
much slower rate of 25 h (29). The same might be true for heart failure is left ventricular assist devices (LVAD) (124,
other ion channels including the potassium and sodium 192), and for arrhythmia management are implantable car-
channels. The precise measurement of channel life cycles at dioverter defibrillators (ICDs) (217). However, the cost of
differential plasma membrane subdomains is limited in the device implantation of either LVAD or ICD and the mor-
literature, which will require a combination of pulse-chase bidity associated with implanted devices are enormous.
experiments with biochemical fractionation and extensive Lack of an accurate understanding of cardiomyocyte health
use of life cell imaging. and recovery potential limits the ability in making critical
decisions with regard to heart transplant priorities and cri-
To provide more insights into ion channel turnover in t-tu- teria for LVAD and ICD implantation (18, 170). New di-
bules versus sarcolemmal membrane, future studies are re- agnostic and prognostic tests that accurately evaluate myo-
quired in primary cardiomyocytes from adult ventricles. cardial health and arrhythmogenic potential at the cellular
The results will also help determine how microdomains in level could significantly improve decision making for LVAD
t-tubules affect ion channel stability compared with non-t- and ICD implantation with improvement in patient care.
tubule plasma membrane. Nevertheless, it is fair to con- New therapeutic tools with the ability to break pathophys-
clude from studies to date that dynamic turnover of ion iological cycling of heart failure progression are acutely
channels at t-tubule membrane is essential for normal func-
needed to help postpone and rescue disease development in
tion of cardiomyocytes, and allows timely regulation of
millions of patients with heart failure.
functional channels under normal physiological conditions.
Furthermore, it has already been established that mutations
For efficient development of new diagnostic, prognostic,
which affect forward trafficking, recycling, and degrada-
and therapeutic tools of human heart failure, a more com-
tion of ion channels, or their interaction with microdomain-
plete understanding of the biology of failing cardiomyo-
organizing proteins such as caveolin-3 and, ankyrin B can
cytes is required. The pathophysiological hallmark of fail-
result in major cardiac diseases (17; review in Ref. 40).
ing cardiomyocytes is weakened calcium transients (74, 75,
128). Normal transients are initiated at cardiac dyads,
VI. ALTERATION OF T-TUBULE STRUCTURE where t-tubule LTCCs (11, 185) are closely associated with
AND FUNCTION IN HEART FAILURE SR RyRs (31, 59, 100, 168) for optimal CICR following
each action potential. Failing calcium transients can be at-
Heart failure is the fastest growing cardiovascular disorder tributed to t-tubule remodeling (132, 133, 224) and dyad
with a high mortality and morbidity affecting more 40 mil- uncoupling (15, 74, 75, 88, 131, 132). T-tubule remodeling

has been considered as a characteristic mark of the transi- sients and electrical instability in failing hearts (FIGURE 10).
tion from hypertrophy to failure (224) and a contributing In zebrafish, morpholino-mediated knockdown of the Bin1
mechanism of heart failure progression (see reviews in Refs. gene induces significant contractile dysfunction and cardio-
19, 20, 61, 80, 99, 132). In addition to the destruction of the myopathy (93). Mice with cardiac conditional Bin1 knock-
gross t-tubule network, protein components at t-tubules out after birth have increased tendency to develop cardio-
including ion channels and signaling molecules are also re- myopathy with either older age or pressure overload (116).
ported to be either reduced or redistributed out of t-tubules. Of the four BIN1 isoforms, cardiac specific BIN1⫹13⫹17
Loss of complex t-tubules in heart failure not only desyn- with inclusion of exons 13 and 17 is the primary BIN1
chronizes CICR and impairs contractility, but also impairs isoform localized to cardiac t-tubules and responsible for
local electrophysiological homeostasis and increases sus- cardiac t-tubule membrane ultrastructure formation (91).
ceptibility to ventricular arrhythmia. Whether aberrant splicing and mutation in cardiac spliced
exons plays a role in cardiomyopathy development remains
Of heart failure-associated molecules, the cardiac t-tubule to be observed. Although the mechanism of reduced BIN1
membrane deformation protein BIN1 is emerging as a cru- expression in heart failure remains to be determined, BIN1-
cial master regulator of healthy t-tubule function, which based microdomain is a potential therapeutic target for the
when reduced can promote heart failure progression (93, development of new heart failure therapies.
116). The role of BIN1 as a cardiac t-tubule adaptor protein
was first noted when constitutive Bin1 knockout in mice In addition to serving as a target for therapeutic develop-
caused perinatal lethality due to cardiomyopathy (150). ment, BIN1 is a potential biomarker of myocardial health
Later it was identified that BIN1 localizes to cardiac t-tu- and reserve. BIN1 is blood available, and plasma BIN1
bules, facilitating microtubule-dependent forward delivery correlates with cardiac health (92), through continuous
of Cav1.2 channels to t-tubule membrane (94). Follow-up turnover of BIN1-microdomains released from the t-tubule
studies from independent research groups identified that membrane. In a cohort of arrhythmogenic right ventricular
BIN1 is transcriptionally decreased in acquired human cardiomyopathy patients, plasma BIN1 decreases in pa-
heart failure (93), as well as multiple animal models of heart tients developing symptomatic heart failure (92). Low
failure (23, 134). Decreased BIN1 level in failing cardiomy- plasma levels of BIN1 correlates with heart failure status
ocytes reduces LTCC surface expression (23, 93, 134), con- and predicts future arrhythmia incidences (92). The re-
tributing to the pathophysiology of diseased calcium tran- cently cloned microdomain forming cBIN1 (91) provides an
Extracellular space Extracellular ions
Actin
RyR
Cytoplasm +Tip BIN1
protein
LTCC
Microtubule
Sarcoplasmic reticulum
FIGURE 10. Reduced cBIN1 microdomains in failing cardiomyocytes. In failing cardiomyocytes with reduced
cBIN1 and cBIN1-induced microdomains, expression of ion channels on the cell surface and the morphology of
t-tubules are altered. As highlighted in the cartoon, changes following reduced BIN1 transcription include 1)
impaired microtubule-dependent targeted delivery of LTCC results in reduced t-tubule surface expression of
LTCCs; 2) disruption of cBIN1 microdomains impairs sufficient LTCC clustering within t-tubules; 3) loss of
dense membrane microfolds and resultant t-tubule dilation cause removal of the protective slow diffusion zone
within t-tubule lumen; and 4) impaired recruitment of RyRs causes accumulation of hyperphosphorylated RyRs
outside dyads, increasing orphaned leaky RyRs.

opportunity to develop a cardiac specific BIN1 blood test to Address for reprint requests and other correspondence:
aid in the diagnosis and management of patients with failing R. M. Shaw, Wasserman Endowed Chair in Cardiology,
hearts. 8700 Beverly Blvd., Davis Bldg. 1016, Los Angeles, CA
90048 (e-mail: robin.shaw@cshs.org).
In the clinical literature, a clear distinction now exists be-
tween heart failure with reduced ejection fraction (HFrEF) GRANTS
and heart failure with preserved ejection fraction (HFpEF),
with HFpEF incidence exceeding that of HFrEF (158). The This work is funded by National Heart, Lung, and Blood
diagnosis of HFpEF is made in patients who present with Institute Grants R00-HL10975 (to T. Hong) and R01-
clinical heart failure despite the absence of left ventricular HL094414 (to R. M. Shaw) and by American Heart
systolic dysfunction. Unlike HFrEF, HFpEF has few avail- Association Grants IRG-16IRG27780031 and BGIA-
able treatment options (142, 158). Newer therapies such as 16BGIA27770151 (to T. Hong) and EIA-13EIA4480016
cardiosphere-derived cell-based treatments are still in the (to R. M. Shaw).
research phase (71). In unpublished studies we have found
that plasma-based cBIN1 decreases in patients with HFpEF DISCLOSURES
just as it does for patients with HFrEF. These types of stud-
ies suggest that future classification of failing hearts will be No conflicts of interest, financial or otherwise, are declared
based less on measures of overall organ function such as by the authors.
ejection fraction, and more on the molecular details of myo-
cyte, and in particular t-tubule, pathophysiology.
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Physiol Rev 97: 227–252, 2017

Published November 23, 2016; doi:10.1152/physrev.00037.2015

CARDIAC T-TUBULE MICROANATOMY

I. INTRODUCTION 227 partmentalize transmembrane ion handling proteins and

0031-9333/17 Copyright © 2017 the American Physiological Society 227

228 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

Intercalated T-tubule Sarcoplasmic reticulum Sarcomere Z-disk

Microtubule Golgi Endoplasmic reticulum Mitochondrion

Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org 229

C TEM D STED E STORM

230 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

BAR PI CLAP MBD SH3

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232 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

Extracellular space Extracellular ions

Microtubule +Tip protein

T-tubule Sarcoplasmic reticulum Myofilament

Adult ventricular cardiomyocyte

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3. Ankyrin B microdomains: NCX/NKA/InsP3 T-tubule membrane is supported by a unique set of peri-

234 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org 235

Although a t-tubule network is a membrane structure com-

236 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org 237

1. LTCC trafficking and clustering to dyads through

238 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

Extracellular space T-tubules

Cytoplasm Cell 1 Cell 2

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GJA1-43k GJA1-32k GJA1-29k GJA1-26k GJA1-20k GJA1-11k GJA1-7k

Transmembrane domain Methionine location corresponding to the AUG start site

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dP-RyR P-RyR Orphaned P-RyR

FIGURE 7. Cartoon of BIN1-dependent recruitment of phosphorylated ryanodine receptors (RyR) to dyads

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Extracellular space Extracellular ions

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