BIN1 Localizes the L-Type Calcium Channel to Cardiac T-Tubules
Citation: Hong T-T, Smyth JW, Gao D, Chu KY, Vogan JM, et al. (
BIN1 Localizes the L-Type Calcium Channel to Cardiac T- Tubules
Ting-Ting Hong 0
James W. Smyth 0
Danchen Gao 0
Kevin Y. Chu 0
Jacob M. Vogan 0
Tina S. Fong 0
Brian C. Jensen 0
Henry M. Colecraft 0
Robin M. Shaw 0
Kenneth R. Chien, Massachusetts General Hospital, United States of America
0 1 Cardiovascular Research Institute, University of California San Francisco , San Francisco , California, United States of America, 2 Department of Medicine, University of California San Francisco , San Francisco , California, United States of America, 3 Department of Physiology, Columbia University , New York, New York , United States of America
The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate Ttubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubulebased Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts.
Funding: This work is funded by the National Institutes of Health grant HL094414. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: Cav1.2, L-type calcium channels; CHF, congestive heart failure; CICR, calcium-induced calcium release; Cx43, Connexin43; EC,
excitationcontraction; NCX1, sodium calcium exchanger 1; SR, sarcoplasmic reticulum; TIRF, total internal reflection fluorescence
The BAR domain superfamily is composed of proteins
involved in endocytosis, organelle biogenesis, cell division, and
cell migration (review in ). As a member of the BAR domain
superfamily, the tubulogenesis membrane scaffolding protein
BIN1 (Amphiphysin 2) is known to induce membrane
invagination [2,3] and initiate tubulogenesis in skeletal muscle cells .
BIN1 deforms the membrane bilayer through interaction
between its N-terminal positively charged BAR domain and
acidic phospholipids within the cell membrane [4,5]. Knowledge
of the role of BIN1 in muscle cells includes evidence of BIN1
distribution on T-tubules of skeletal myocytes  and that
constitutive knockdown of BIN1 in mice is perinatal lethal, with
pathology revealing a hypertrophic dilated cardiomyopathy [6,7].
However, despite these findings, little is known of the functional
role of BIN1 in cardiomyocytes.
Since BIN1 knockdown results in cardiomyopathy , it is
possible that BIN1 may play a role in regulating the cardiac
calcium transient. During each heartbeat, calcium release from
intracellular stores is achieved when trans-sarcolemmal calcium
activates the ryanodine release channels on the sarcoplasmic
reticulum (SR) . The initial calcium influx occurs primarily
through the L-type calcium channels with Cav1.2 as the
poreforming subunit. Trans-sarcolemmal calcium entry and activation
of ryanodine receptors is a local phenomenon and, in
cardiomyocytes, sarcolemmal Cav1.2 channels occur within 15 nm of their
respective ryanodine receptors on the SR . A major function of
T-tubule invaginations of the sarcolemma, which are enriched
with Cav1.2 channels [10,11], is to bring the channels into close
Calcium plays a primary role in regulating heart function.
During each heartbeat, calcium ions cross the membrane
of individual cardiac muscle cells and trigger a rapid
increase of calcium within the cell (called the calcium
transient). Calcium causes the muscle cells to contract and
determines the strength of the overall heartbeat. Each
cardiac muscle cell has many small tubular-like membrane
invaginations known as T-tubules where calcium channels
localize, allowing calcium ions to enter and immediately
encounter intracellular calcium release organelles. While
this organization is well described, it is not known how
calcium channels localize to T-tubule membrane. Here we
show that in human and mouse heart cells, a membrane
scaffolding protein known as BIN1 is localized together
with calcium channels at T-tubules. Using high-resolution
live cell microscopy, we found that microtubules, which
are necessary for calcium channel delivery to the
membrane, are also tethered by BIN1. Loss of BIN1 in
cardiac cells impairs delivery of calcium channels to the
membrane and diminishes the intracellular calcium
transient. According to this model, microtubules function
as highways that carry newly synthesized calcium channels
to BIN1-containing membrane. Once tethered to T-tubules
by BIN1, the microtubules can deliver their calcium
channel cargo. We postulate that this calcium channel
delivery pathway is important to the regulation of cardiac
calcium signaling and beat-to-beat cardiac function.
proximity of the ryanodine receptors, amplifying sarcolemmal
calcium entry to a large calcium release from the SR. This process,
which is known as calcium-induced calcium release (CICR) , is
essential to each heartbeat and links electrical excitation of the
myocyte and local calcium entry to its mechanical contraction.
The mechanism for Cav1.2 localization to T-tubules remains
It is possible that locally enriched BIN1 may assist in the
delivery of Cav1.2 channels in a manner similar to the role of
adherens junctions in aided delivery of Connexin43 (Cx43)
hemichannels to intercalated discs , a highly efficient
trafficking pathway for polarized protein distribution. Therefore,
depletion of BIN1 at T-tubule membrane after knockdown could
result in mislocalization of Cav1.2, causing inefficient
excitationcontraction (EC) coupling and lethal cardiomyopathy. Supporting
evidence for this Cav1.2 localization hypothesis is that another
BAR domain containing protein, endophilin, has been shown to
complex with Cav1.2 at the plasma membrane . Furthermore,
BIN1 has been shown to interact not only with cortical actin
[15,16] but also with a microtubule plus end tracking protein .
These data indicate that BIN1 might be closely associated with
growing microtubules, a key component of the trafficking
machinery for targeted delivery.
In this study, we provide data supporting a role for BIN1 in
tethering microtubules for direct delivery of L-type calcium
channels to cardiac T-tubules. We observed that in both human
and mouse cardiomyocytes, BIN1 and Cav1.2 colocalize at
cardiac T-tubules (by fluorescence and electron microscopy
immunogold labeling) and co-immunoprecipitate. Regarding
delivery to T-tubules, we found that BIN1 tethers dynamic
microtubules and forward trafficking of Cav1.2 channels is
microtubule dependent. In reductionist atrial myocyte and
nonmyocyte cell systems, BIN1 is sufficient to form membrane
invaginations and distribute Cav1.2 to these BIN1-containing
membrane regions (visualized by total internal reflection
microscopy, TIRFm). The delivery results in non-myocyte cells suggest
that Cav1.2 delivery to BIN1 is independent of other
myocytespecific organelles and proteins. To rule out that the sarcolemmal
invaginations themselves and not BIN1 are sufficient for Cav1.2
delivery, we created C-terminal truncated BIN1, which fails to
attract Cav1.2 yet still inducing membrane invagination. In
isolated primary mouse ventricular cardiomyocytes, disruption
of this delivery mechanism by BIN1 knockdown results in less
surface expression of Cav1.2 and abnormal calcium transient
Our findings indicate that the membrane curvature protein
BIN1 can form membrane invaginations and is localized to
cardiac T-tubules, providing an anchor for microtubules that
allows targeted delivery of Cav1.2 channels and regulation of the
cardiac calcium transient. A role of BIN1 in facilitating
microtubule-based antegrade delivery of membrane protein traffic
adds an important facet to the multifunctional BAR domain
family. Furthermore, our findings suggest that microtubule-based
delivery of Cav1.2 to BIN1 is significant to cardiac calcium
BIN1 Is Distributed along Cardiac T-Tubules and
Colocalizes with Cav1.2
To understand the cellular distribution of BIN1 in mammalian
cardiomyocytes, we dissociated non-failing human cardiomyocytes
from freshly explanted human hearts with normal left ventricular
function, as well as normal adult mouse cardiomyocytes. After
fluorescence immunostaining, the cardiomyocytes were imaged at
Z-depth increments of 0.1 mm with a spinning disc confocal
microscope and viewed in two-dimensional frame views along the
longitudinal axis. BIN1 has a nuclear localization, as previously
reported, in embryonic hearts (Figure S1) , but elsewhere in the
cardiomyocyte, a typical T-tubule distribution pattern of BIN1
emerges that is similar to Cav1.2 distribution (Figure 1A, first row).
Representative fluorescence intensity profiles along the
longitudinal axis of cardiomyocytes are in the second row of Figure 1A.
Note that there is a fluorescence signal peak approximately every
2 mm, which corresponds to the T-tubule distribution of the
protein. Power spectrum analysis  confirms that the
fundamental periodicity of Cav1.2 is 2 mm (Figure 1A, third row), which
is consistent with previously reported cardiac T-tubule intervals
[18,19]. BIN1 shows the same spatial periodicity as Cav1.2 in
human and mouse (Figure 1A) cardiomyocytes. Cx43, which
localizes at intercalated discs at the longitudinal ends of
cardiomyocytes, does not have the same spatial periodicity and
serves as a negative control (Figure S2). The data of Figure 1A
indicate that BIN1 is localized along T-tubules in cardiomyocytes.
Next, we quantified colocalization between Cav1.2 and BIN1.
In Figure 1B, immunolabeling of BIN1 (green) and Cav1.2 (red) is
shown in subsections of both human and mouse cardiomyocytes.
Full cardiomyocyte views of colocalization between BIN1 and
Cav1.2 are shown in Figure S3. The data indicate that BIN1
significantly colocalizes with Cav1.2, primarily at T-tubules. For
negative control studies, Cav1.2 does not have significant
colocalization with Cx43 (Figure S4). To confirm spatial
coincidence, we used transmission electron microscopy with dual
immunogold labeling to identify Cav1.2 and BIN1 on T-tubule
ultrastructures in adult mouse cardiomyocytes. Results in
Figure 1C (left panel) indicate that BIN1 (small 10 nm dots) and
Cav1.2 (large 15 nm dots) are enriched and occur within 10
50 nm of each other at T-tubular membrane structures.
BIN1 Tethers Dynamic Microtubules Involved in
Antegrade Trafficking of Cav1.2
The data in Figure 1C indicate close approximation of Cav1.2
and BIN1 in isolated cardiomyocytes but do not reveal how the
proteins achieve such localization. There is significant support for
membrane ion channel delivery occurring via microtubules
[13,20,21]. To address whether BIN1 serves as a microtubule
anchoring site to allow Cav1.2 delivery, we first studied microtubule
behavior in the vicinity of BIN1 in HeLa cells, which are permissive
to high-resolution imaging. HeLa cells were transfected with
atubulin-GFP and BIN1-mCherry. Introduction of exogenous BIN1
forms membrane invaginations as previously reported in other
nonmyocyte cell types . Twenty-four hours post-transfection,
microtubule dynamics were recorded with spinning disc confocal
microscopy for 2 min with a frame rate of 1 s. As seen in the
enlarged panel of the overlay between BIN1 (red) and microtubules
(black lines) in Figure 2A, microtubules tether at BIN1 structures.
Three representative microtubule travel paths involving a
microtubule that remains at BIN1 (MT1), a microtubule that departs
BIN1 (MT2), and a microtubule that approaches BIN1 (MT3) are
also indicated in green. For each of these three microtubules, the
distance between the microtubule tip and the center of the closest
BIN1 structure is plotted over time in Figure 2B. In each graph, the
distance within 0.2 mm of the respective BIN1 structure is
highlighted in red dotted lines. MT1 has paused at BIN1 structure
for the whole 2 min imaging window and has relatively little
movement. However, MT2 pauses and hovers at BIN1 and, upon
leaving, picks up velocity, while MT3 approaches BIN1 with high
Figure 2. BIN1 tethers dynamic microtubules. (A) HeLa cells were transfected with a-Tubulin-GFP and BIN1-mCherry. The overlay pictures of
BIN1 (red) and microtubules (black) are shown in the left panel. The right image is an enlarged subsection of the left image. Three microtubule travel
paths (MT1, MT2, and MT3) are also highlighted in green in the subsection. (B) Graphs of each microtubule travel path. BIN1 edge (within 0.2 mm of
BIN1 structure) is highlighted with a red dotted line in each graph.
At BIN1 (n = 20)
Away from BIN1 (n = 19)
Table 1 contains the overall travel velocity, growth, and shortening velocities, as well as pausing events of microtubules whose tips are within 0.2 microns of, or away
from, BIN1 structures. n is number of events. Data are from 15 microtubules in four different cells.
velocity before it slows down as it comes into contact with BIN1.
The dynamic movements of MT1, MT2, and MT3 are shown in
Video S1. In addition, the overall microtubule dynamics tabulated
from 15 microtubules of four individual cells are presented in
Table 1. These data indicate that overall tip velocity is 56 faster
(0.15 mm/s versus 0.03 mm/s) when the microtubules are not in the
proximity of BIN1. This increased overall velocity consists of not
only faster growth and shortening velocities but also less frequent
pauses. The data from Figure 2 and Table 1 suggest that
microtubules are tethered by BIN1 structures.
To evaluate if microtubules are involved in antegrade trafficking
of Cav1.2 channels, we exposed live primary adult ventricular
cardiomyocytes to the microtubule disruptor nocodazole in the
presence of dynasore, a specific dynamin GTPase inhibitor that
blocks endocytosis . Expression of surface membrane-bound
Cav1.2 was assayed by cell surface biotinylation (Figure 3A).
Dynasore treatment alone increases surface expression of Cav1.2,
indicating inhibition of Cav1.2 endocytosis. In the presence of
both dynasore and nocodazole, Cav1.2 surface expression
progressively decreases, further suggesting that microtubule
disruption reduces forward trafficking of Cav1.2 to the plasma
membrane. To confirm that delivery of Cav1.2 to T-tubules is
microtubule dependent, the cellular distribution of Cav1.2 in
cardiomyocytes subjected to nocodazole was studied by
immunoconfocal microscopy. As seen in Figure S5, nocodazole decreases
Cav1.2 surface expression not only at T-tubules (Figure S5,
bottom right) but also at global sarcolemma containing
non-Ttubule membrane (Figure S5, bottom left). The total cellular
protein expression level of Cav1.2 is not changed by nocodazole
(Western blot in Figure S5, top panel). Microtubule-dependent
trafficking of Cav1.2 is further supported by the localization of
Cav1.2 vesicles along the microtubule network in the vicinity of a
T-tubule in adult mouse cardiomyocytes (Figure 3B, top panel).
To better visualize microtubules and Cav1.2, we used the
cardiomyocyte-derived HL-1 cell line that has a morphology
amenable to high-resolution imaging  and find that Cav1.2
distributes along the microtubule network (Figure 3B, bottom
panel). Comparable biotinylation results confirm
microtubuledependent surface expression of Cav1.2 in HL-1 cells (Figure S6).
From the data in Figures 13, it appears that BIN1 is enriched along
cardiac T-tubules and closely associated with Cav1.2. Furthermore,
BIN1 tethers to plasma membrane dynamic microtubules, which
deliver Cav1.2 to the plasma membrane. Therefore, it is possible that
BIN1 is a T-tubule anchor site for targeted delivery of Cav1.2 through
the interaction between BIN1 and growing microtubules.
Cav1.2 Concentrates at BIN1-Induced Membrane
To test the exclusivity of the relationship between Cav1.2 and
BIN1, we explored whether Cav1.2 could be targeted to
exogenous BIN1-induced membrane invaginations in cell lines
lacking a developed T-tubule system. HL-1 cells are myocytes that
express endogenous Cav1.2 but do not have a developed T-tubule
system. Introduction of exogenous BIN1 generates membrane
invaginations of cell membrane that appear as linear streaks , as
seen in Figure 4A (green, with Cav1.2 in red). As indicated by the
structures near the arrows in the right panel of Figure 4A, Cav1.2
localizes to exogenous BIN1, just as Cav1.2 localizes to
endogenous BIN1 in primary cardiomyocytes seen in Figure 1.
To confirm that membrane delivery of Cav1.2 to BIN1 can occur
in non-myocyte cells, we evaluated surface expression patterns of
exogenous Cav1.2 in HeLa cells expressing exogenous BIN1. In
order to resolve BIN1 structures at the level of plasma membrane,
we used TIRFm, which limits the imaging depth to within 50
100 nm. Using Cav1.2 and BIN1 tagged with spectrally distinct
fluorophores, we performed a brief time lapse capture, with
representative results shown in Figure 4B. The data indicate that
BIN1-induced structures (green) attract surface Cav1.2 (red),
causing local enrichment of calcium channel. Thereby, in the
absence of other myocyte structures as well as the absence of
endogenous Cav1.2, ectopic expression of BIN1 is sufficient to
concentrate surface Cav1.2. The possibility of close biochemical
association between BIN1 and Cav1.2 in HeLa cells is further
supported by co-immunoprecipitation of V5-tagged BIN1 and
Cav1.2 (Figure 4B). In summary, we find that microtubule-based
delivery of Cav1.2 to tubular membrane invaginations is BIN1
dependent and is independent of other myocyte-specific structures
and proteins (Figure 4C).
Cav1.2 Targeting Requires BIN1, Not Membrane
To confirm that it is specifically BIN1, and not the
BIN1induced membrane invaginations, that localizes Cav1.2, we used a
truncation mutant of BIN1. Full-length BIN1 (1-454 aa) has an
Nterminal BAR domain followed by a coiled-coil linkage domain
and a C-terminal SH3 domain (Figure 5A) [4,24]. Following
precedent , we created a C-terminal truncated BIN1-BAR*
(1-282 aa), which retains the ability to induce membrane
invagination (the electron microscopy membrane structures are
shown in Figure 5B). However, BIN1-BAR* loses the ability to
attract endogenous Cav1.2 to the nascent membrane
invaginations such as those in HL-1 cells (Figure 5C). With full-length
BIN1 (top row), endogenous Cav1.2 is distributed along BIN1
structures. In contrast, Cav1.2 (red) has poor colocalization with
BIN1 structures (green) in cells transfected with BIN1-BAR*
(bottom panel). The effect of full-length BIN1 and BIN1-BAR* on
Cav1.2 surface targeting was further tested by a biochemical
surface biotinylation assay. As in Figure 6, unlike BIN1-BAR*,
full-length BIN1 has greater surface expression of Cav1.2. Thus,
targeting of Cav1.2 to membrane invaginations requires
fulllength BIN1. It appears that BIN1 recruitment of Cav1.2 involves
a domain distinct from that which induces membrane curvature.
To determine specificity of BIN1 to Cav1.2, we repeated the
surface biotinylation assay for the sodium calcium exchanger 1
(NCX1), which is also a T-tubule localized channel. As seen in
Figure S7, BIN1 fails to increase surface expression of NCX1,
indicating that BIN1-based delivery has specificity for Cav1.2.
In Mouse Cardiomyocytes, BIN1 Knockdown Delays
We then investigated whether disruption of such a
T-tubuletargeting mechanism of Cav1.2 impacts cardiomyocyte function.
Although T-tubules are only partially developed in freshly
dissociated neonatal cardiomyocytes [25,26], earlier studies by
electron microscopy show T-tubules develop after 3 days
differFigure 4. Cav1.2 is targeted to BIN1-induced membrane structures. (A) Deconvolution of wide-field image (1006) of BIN1 transfected HL-1
cells indicates endogenous Cav1.2 (red) colocalizes with exogenous BIN1 (green) (scale bar: 5 mm). (B) TIRFm images of a HeLa cell transfected with
Cav1.2-GFP (red) and BIN1-mCherry (green) reveal colocalization between BIN1 and Cav1.2 at the cell periphery (scale bar: 5 mm). This panel also
includes co-immunoprecipitation between overexpressed BIN1-V5 (IP) and Cav1.2 (IB) in HeLa cells. (C) A schematic of dynamic microtubules
delivering Cav1.2 to BIN1 at T-tubules.
Figure 5. Cav1.2 is targeted to BIN1, not membrane invaginations. (A) Domain map of wild-type BIN1 (BIN1). BIN1-BAR* (1-282 aa) contains
the BAR domain and the sequence upstream of the coiled-coil region that is necessary for inducing membrane invagination. (B) Electron microscopy
images indicate that BIN1 and BIN1-BAR* form similar membrane invaginations (dark linear tubules). (C) Deconvolved wide-field image of HL-1 cells
transfected with BIN1 or BIN1-BAR*(1-282 aa). Co-staining between endogenous Cav1.2 (red) with transfected exogenous BIN1 or BIN1-BAR* (green)
indicates that Cav1.2 localizes to BIN1 structures but not BIN1-BAR* structures (scale bar: 5 mm).
entiation in culture  along with redistribution of
Z-lineassociated cytoskeleton proteins for Z-line organization .
Recent studies also find that in cultured differentiated neonatal
cardiomyocytes, the dihydropyridine receptor  and other
components of the calcium-release and uptake machinery , as
well as other T-tubule proteins , develop a typical T-tubule
staining pattern. Similarly, we observed T-tubule-type staining in
cells dissociated at postnatal day three or four and allowed to
differentiate in culture for a week. These structures were enriched
with both Cav1.2 and BIN1 (Figure S8A). Furthermore, BIN1
mRNA expression in postnatal mouse heart tissue is similar to that
in adult heart (Figure S8B). Using this differentiated mouse
cardiomyocyte population, BIN1 siRNA successfully decreases
BIN1 expression by 80%, as assayed by Western blot in Figure 7A.
As a result of BIN1 knockdown, surface Cav1.2 is reduced by
45%, although the total cellular protein expression of Cav1.2
remains similar (Figure 7B). To assay the effect on cardiomyocyte
calcium transients, we loaded the cells with a fluo 4-AM and
imaged with a wide-field epifluorescence microscope. As seen in
Figure 7C, loss of BIN1 results in a significant slowing of calcium
transient development, indicating reduced CICR. The slowing of
calcium transient development is quantified by measuring the time
to reach 50% of peak calcium concentration (T1/2 max). BIN1
knockdown delayed T1/2 max by 40% (bar graph, Figure 7C).
The data in Figure 7 indicate that knockdown of BIN1 reduces the
surface expression of Cav1.2, impairing the intracellular cardiac
transient, and that BIN1 is necessary to maintain normal calcium
signaling in the heart.
Figure 6. Full-length BIN1 causes Cav1.2 surface expression. Surface biotinylation of Cav1.2 in HL-1 cells transfected with either BIN1 or
BIN1BAR* reveals that full-length BIN1 is required to cause surface expression of Cav1.2 (* p,0.05, Students t test).
The primary finding of this study is the identification of a novel
role for BIN1 as a T-tubule anchoring protein accepting antegrade
delivery of Cav1.2. Immunocytochemical staining indicates that
BIN1 colocalizes with Cav1.2 along T-tubules in primary adult
human and mouse cardiomyocytes (Figure 1). Dual immunogold
transmission electron microscopy images reveal that Cav1.2 and
BIN1, which co-immunoprecipitate (Figure 4), cluster together
within ,1050 nm on T-tubules (Figure 1C). Regarding delivery
of Cav1.2 to T-tubules, there is significant support for membrane
ion channel delivery occurring via microtubules [13,20,21]. In
exploring the forward trafficking mechanism of Cav1.2, we find
that microtubules are required for the delivery of Cav1.2 (Figure 3)
and that BIN1 anchors microtubules (Figure 2, Table 1), which
can provide offloading of Cav1.2-containing vesicles to T-tubule
BIN1 is a member of the BAR domain containing protein
family, which has a role in membrane bilayer deformation at
endocytic sites through interaction between their N-terminal
positively charged BAR domains and acidic phospholipids within
cell membrane . Fluorescence and electron microscopy reveal
that a human BIN1 construct can induce enormous membrane
invaginations in both non-T-tubule-forming atrial HL-1 cells and
non-cardiac HeLa cells (Figures 4A, 4B, 5B), as previously
reported in other cell types . If BIN1 at cardiac T-tubules is
closely associated with Cav1.2 (Figure 1) and dynamic
microtubules (Figure 2), it is possible that BIN1 alone is sufficient to target
microtubule-transported Cav1.2. In myocyte HL-1 cells,
overexpression of exogenous BIN1 changes the cellular distribution of
endogenous Cav1.2 and relocalizes them to nascent BIN1-induced
membrane invaginations (Figure 4A). Furthermore, loss of BIN1 in
cardiomyocytes reduces surface expression of Cav1.2 (Figure 7B).
Such delivery is not myocyte dependent. In HeLa cells, which are
devoid of the cardiac-specific cellular ultrastructures and
machinery, overexpression of BIN1 is sufficient to localize exogenous
Cav1.2 to the cell periphery on BIN1 membrane structures as
resolved by simultaneous dual-color TIRFm (Figure 4B).
Coimmunoprecipitation of BIN1 and Cav1.2 (Figure 4B) further
indicates that they are present in the same protein complex. This
physical association between BIN1 and Cav1.2 further supports
the model that BIN1 serves as a membrane anchor site for Cav1.2
T-tubules are a well-organized membrane structure in which it
is unknown how the T-tubule-related proteins localize there,
specifically for Cav1.2. How could we then exclude the possibility
that the membrane invaginations alone are sufficient to cause
Cav1.2 delivery to T-tubules independent of BIN1? As previously
established , the extended BAR domain (BAR*, amino acid 1
282, Figure 5A) of BIN1 is sufficient for inducing membrane
invagination (Figure 5B), despite the absence of other domains
responsible for protein-protein interaction. Distinct from
fulllength BIN1, BIN1-BAR* neither redistributes Cav1.2 (Figure 5C)
nor causes Cav1.2 surface expression (Figure 6). It appears that
Figure 7. BIN1 knockdown delays calcium transient development in mouse cardiomyocytes. (A) Western blot indicates an 80%
knockdown of BIN1 protein by siRNA in differentiated mouse cardiomyocytes. (B) Surface biotinylation of Cav1.2 in these primary cardiomyocytes
indicates a 45% reduction of surface Cav1.2 after BIN1 knockdown. (C) Live cell calcium imaging in differentiated cardiomyocytes indicates that BIN1
knockdown also delays calcium transient development in these cells. Average time to 50% maximal fluorescence intensity (T1/2 max) of calcium
transient is presented in the left panel (* p,0.05, ** p,0.01, Students t test).
BIN1 recruitment of Cav1.2 involves a domain distinct from the
one that induces membrane curvature, as suggested by endophilin
binding to Cav1.2 in its non-BAR coiled-coil region .
The BAR domain superfamily has previously been associated
with anchoring cortical actin at the plasma membrane [15,16].
This study introduces microtubule anchoring as well (Figure 2).
There may be a general role for BAR domain proteins in allowing
antegrade trafficking and localization of membrane-bound
proteins. Furthermore, the mechanism of targeting Cav1.2 to
Ttubules may be responsible for diseases associated with genetic
BIN1 dysfunction. In mice, BIN1 knockout causes perinatal lethal
cardiomyopathy , and a mutation in the same 2q14-22 locus of
BIN1 is associated with familial cardiomyopathy in humans .
Loss of function mutations in BIN1 also results in centronuclear
peripheral myopathy  characterized by muscle weakness,
which could be explained by calcium dysregulation. Future studies
will be required to investigate whether calcium channel trafficking
and localization are altered in these diseases.
With regard to cardiac myocytes, our findings constitute a new
understanding of calcium channel regulation. In order to allow
trans-sarcolemmal calcium to reach the intracellular ryanodine
receptors, Cav1.2 channels must be localized at T-tubules . It
has been estimated that T-tubule calcium channels contribute
80%90% of the total cellular calcium current [10,11].
Furthermore, Cav1.2 experiences a high turnover, with pulse chase
experiments indicating a half-life as short as 3.5 h . The need
for specific localization with a rapid turnover implicates that
channel delivery is an important and highly regulated aspect of
Cav1.2 channel function. Indeed, our data indicate that T-tubule
targeting of Cav1.2 by BIN1 is critical in calcium handling and
regulation in cardiomyocytes. As seen in Figure 7, loss of BIN1
reduces surface Cav1.2 and delays calcium transient development
in primary cardiomyocytes. The data indicate that BIN1 functions
as a T-tubule-membrane-anchoring site for microtubules to deliver
Cav1.2, thereby ensuring proper control of cardiac EC coupling.
Moreover, the mechanistic understanding of Cav1.2 trafficking
to T-tubules by our current study not only provides insight into
calcium regulation in normal hearts but also has significant
implications in the pathogenesis of diseases with altered calcium
dynamics such as congestive heart failure (CHF). In failing heart,
the intracellular calcium transient of ventricular cardiomyocytes
has a low amplitude and slow decline , resulting in
compromised contraction . Multiple factors downstream of
calcium entry through Cav1.2 have been identified in failing
muscle that contribute to changes in the calcium transient,
including dysfunction in calcium removal [38,39] and, more
recently, phosphorylation and perturbation of the ryanodine
release channels [40,41]. There have also been reports that
dyssynchronous CICR may exist in failing cardiomyocytes and
contribute to defective EC-coupling gain in failing heart [42,43].
Since localization of L-type calcium channels is critical for
synchronous CICR, loss or mislocalization of these channels in
the local microenvironment might lead to defective CICR and
abnormal heart function. In fact, human CHF has reduced L-type
calcium channel density in the sarcolemma , and a canine
model of heart failure is associated with remodeling of both
Cav1.2 distribution and T-tubule structure . In this study, we
found that BIN1-based microtubule targeting affects Cav1.2
localization and intracellular calcium dynamics (Figure 7). It will
be interesting in future studies to explore the role of BIN1
regulation in heart failure.
Materials and Methods
Plasmids, Cell Culture, and Transfection
Human BIN1 (Isotype 8) cDNA was obtained from Origene.
Full-length BIN1-8(1-454 aa) and BIN1-BAR*(1-282 aa) were
then amplified and cloned into pDONR/Zeo (Invitrogen) using
Gateway BP cloning to generate entry clones. The genes were
subsequently inserted into pDest-eGFP-N1, pDest-mCherry-N1
(converted vectors originally from Clontech), and
pcDNA3.2-V5Dest by Gateway LR cloning. Human Cav1.2 was obtained from
Origene. Human b2b and rabbit a2d1 were generously provided
by Dr. Michael Sangunetti. N-terminal GFP-Cav1.2 was
generously provided by Dr. Kurt Beam, and C-terminal Cav1.2-GFP
was described previously . Non-targeting and BIN1-specific
siRNA were obtained from Dharmacon.
HeLa cells and mouse atrial HL-1 cells were cultured in
DMEM and Claycomb medium under standard mammalian cell
conditions. FuGene 6 (Roche) was used for cDNA transfections in
HeLa cells. Lipofectamine (Invitrogen) was used for cDNA
transfections in HL-1 cells.
Dissociated cardiomyocytes were allowed to attach to
lamininprecoated glass coverslips before fixation. For all
immunocytochemistry, cells were fixed in methanol at 220uC for 5 min. For
immunohistochemistry, cryosections were fixed in ice-cold acetone
for 10 min. After fixation, cardiomyocytes were permeablized and
blocked with 0.5% Triton X-100 (Sigma) and 5% NGS in PBS for
1 h at room temperature. For BIN1 and Cav1.2 staining, the cells
were incubated with mouse anti-BIN1 (1:50, Sigma) and rabbit
anti-Cav1.2 (1:50, Alomone) overnight at 4uC. Similar protocol
without permeablization was used for BIN1 and Cav1.2 staining in
myocardium cryosections. For co-staining of Cav1.2 and a-tubulin
in HL-1 cells, after fixation, the cells were permeablized with 0.1%
Triton X-100 for 15 min and blocked with 5% NGS for 1 h. The
cells were then incubated with rabbit anti-Cav1.2 (1:50, Alomone)
overnight at 4uC followed by mouse monoclonal to a-Tubulin
(1:500, Sigma) for 1 h at room temperature. After several washes
with PBS post-primary antibody incubation, cells were then
incubated with goat anti-mouse and -rabbit IgG conjugated to
AlexaFluor 488 and 555, respectively. Cells were then fixed and
mounted with DAPI containing ProLong gold.
Wide-Field Epifluorescence, TIRF, and Spinning Disc
All imaging was performed on a Nikon Eclipse Ti microscope
with a 1006 1.49 NA TIRF objective and NIS Elements software.
Deconvolution of images was performed using Autoquant software
(Media Cybernetics). High-resolution cardiomyocyte images were
obtained by a spinning disc confocal unit (Yokogawa CSU10) with
DPSS lasers (486, 561) generated from laser merge module 5
(Spectral applied research, CA) and captured by a high-resolution
Cool SNAP HQ2 camera (Photometrics). Multiple wavelength
TIRF was achieved with Dual-View emission splitter (Optical
Insights). High-sensitive Cascade II 512 camera (Photometrics)
was used for TIRF image capture.
For BIN1 and Cav1.2 distribution, isolated human and mouse
cardiomyocytes were imaged at Z-depth increments of 0.1 mm and
reconstructed to generate three-dimensional volume views and
frame view along the longitudinal axis using NIS Element
Software. To access Cav1.2 and BIN1 colocalization by TIRFm,
HeLa cells were plated overnight and co-transfected with
pDestBIN1-mCherry, and Cav1.2-GFP along with b2b and a2d1. Dual
channel TIRF time lapse sequences of 1 min were acquired at an
exposure of 200 ms per image at a rate of 1 frame per second.
After acquisition, the total 61 frames were z-projected into one
frame using ImageJ (NIH).
For live-cell imaging of microtubule behavior by spinning disc
confocal microscopy, Hela cells were plated and co-transfected
with pDest-BIN1-mCherry and a-tubulin-GFP. Time lapse
sequence for a-tubulin was acquired at a continuous rate of 1 s
with 400 ms exposure per frame. To confirm the similar BIN1
expression pattern, BIN1 images were taken both before and after
tubulin time lapse sequence. The tubulin-GFP particle paths were
manually traced and analyzed for travel velocity and pause event
in the time sequence using MTrackJ Plugin in ImageJ.
Microtubule at BIN1 is considered when the microtubule tip is
within 0.2 mm of the closet BIN1 edge.
For calcium imaging in neonatal mouse cardiomyocytes,
cardiomyocytes were loaded with a cell permeable calcium dye
4-AM in calcium-free HBSS (Gibco) for 15 min and imaged in
regular HBSS (Gibco) with a 206TIRF objective with a wide-field
epifluorescence microscopy. Live images were captured by a
highsensitive Cascade II 512 camera at a frame rate of 64 ms for 20 s.
For electron microscopy membrane ultrastructure, cells were
fixed in Karnovskys fixative (1% paraformaldehyde / 3%
Glutaraldehyde in 0.1 M Sodium cacodylate buffer, pH 7.4) at
room temperature for 30 min before being stored at 4uC. The
method for the membrane ultrastructure study was previously
described [47,48]. Briefly, the fixed cells were then post-fixed in
OsO4 (2% OsO4 + 1.5% potassium ferrocyanide, Sigma) and
stained en bloc with 1% tannic acid (Sigma), uranyl acetate (EM
Science) before being dehydrated in ethanol, cleared in propyline
oxide, and embedded in eponate 12 (Ted Pella Co.). Finally, cells
were sectioned and stained with uranyl acetate and Reynolds
Lead to enhance contrast and were examined under Philips
Tecnai 10 electron microscope (Eidhoven).
For immunolabeling, mouse cardiomyocyte suspension was
fixed in 2% paraformaldehyde / 0.1% glutaraldehyde in 0.1 M
cacodylate buffer pH7.4 at room temperature for ,23 h. An
established procedure [49,50] was used for immunogold labeling
of mouse cardiomyocytes. Briefly, the fixed samples were
cryoprotected with PVP/sucrose (20% polyvinyl pyrrolidone
[Sigma] in 2.3 M sucrose) overnight and frozen in liquid nitrogen
before being cut into thin sections with Leica Ultracut UCT with
EMFCS attachment (Leica Microsystems Inc.). Sections were
treated with 0.2% glycine, blocked with 2% BSA/gelatin in PBS,
pH 7.4, incubated with mouse anti-BIN1 (1:2, Sigma) and rabbit
anti-Cav1.2 (1:2, Alomone) diluted with blocking solution
overnight at room temperature (controls were done with normal
mouse serum), and incubated with 10 nm immunogold conjugated
anti-mouse (1:25) and 15 nm immunogold conjugated goat
antirabbit (1:50) secondary antibodies for 30 min. The sections were
then stained with oxalate uranyl acetate and embedded in 1.5%
methyl cellulose (Sigma) and 0.3% aqueous uranyl acetate (Ted
Pella Inc.). Colocalization between Cav1.2 and BIN1 was
examined in a Philips Tecnai 10 electron microscope (Eidhoven).
With the approval of the University of CaliforniaSan Francisco
(UCSF) Committee for Human Research, we obtained tissue from
organ donors whose hearts were not transplanted. The California
Transplant Donor Network (CTDN) provided the unused donor
hearts and obtained informed consent for their use from the next
of kin. All the mouse work was approved by UCSF Committee for
Animal Research. All procedures were in accordance with UCSF
animal research and care protocols.
Human Tissue Collection and Cardiomyocytes Isolation
After immediate perfusion with cold cardioplegia, full-thickness
samples from left ventricular free wall were cleaned rapidly of all
epicardial fat and snap frozen into liquid nitrogen for later protein
and mRNA analysis. More sections were embedded in OCT
medium and frozen in liquid N2-chilled isopentane for
immunohistochemistry. For cardiomyocytes isolation, ventricular free wall
samples were cut into ,1 mm3 sections for digestion with
prewarmed collagenase II (2 mg/ml, Worthington) at 37uC in
calcium-free KHB solution (134 mM NaCl, 11 mM Glucose,
10 mM Hepes, 4 mM KCl, 1.2 mM MgSO4, 1.2 mM Na2HPO4,
10 mM BDM, 0.5 mg/ml BSA, Ph 7.4)  with modification of
a previously reported method . Dissociated cardiomyocytes
were allowed to attach to laminin-precoated glass coverslips before
fixation for immunocytochemistry.
Isolation and Culture of Adult Mouse Cardiomyocytes
Mouse ventricular myocytes were isolated from male adult C6/
Black mouse (,812 wk; Charles River) after dissociation with
collagenase II (2 mg/ml, Worthington) with a previously described
method . For surface biotinylation experiments,
cardiomyocytes were attached to laminin-precoated culture dishes and
cultured in primary cardiomyocyte medium (ScienCell) in 37uC
and 5% CO2 incubator. The cells were treated with vehicle
(DMSO, 1:2000) overnight (16 h) before the replacement with
control medium (containing DMSO, 1:2000) or medium
containing 20 mM dynasore with or without 30 mM nocodazole for 2 h.
For 18 h nocodazole treatment, cardiomyocytes were cultured in
medium containing 30 mM nocodazole overnight (16 h) before the
replacement of medium containing dynasore + nocodazole for
another 2 h.
Isolation and Differentiation of Mouse Cardiomyocytes
Timed pregnant mice were ordered from Charles River at
E1617. Primary mouse neonatal cardiomyocytes were isolated from
p3/4 C57BL/6 mice and maintained in F12/DMEM 50/50
(Invitrogen) supplemented with 2% FBS,
Insulin-transferrinsodium selenite media supplement, 10 mM
5-Bromo-29-deocyuridine, 20 mM Cytosine b-D-arabinofuranoside (Sigma), and
100 mg/ml Primocin (Amaxa). Cells were maintained in a
humidified atmosphere of 5% CO2 at 37uC. Cardiomyocytes
were allowed for differentiation in culture for about a week before
surface biotinylation and calcium-imaging experiments. After 3 to
4 d in culture, the cells were transfected with 125 nM control or
BIN1 siRNA (Dharmacon), which was repeated 24 h later. Three
days after the first dose of siRNA, surface biotinylation
experiments and calcium imaging were studied in these cells.
Surface Biotinylation of Cav1.2 and NCX1
After treatment, the cells were quickly washed and incubated
with ice-cold 1 mg/ml High Capacity Neutraavidin Agarose Resin
(Pierce) for 25 min. After 2 6 5 min quenching of unbound biotin
with 100 mM glycine, cells were washed and lysed in RIPA buffer
(50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton
X-100, 1% sodium deoxycholate, 2 mM NaF, 200 mM Na3VO4)
supplemented with Complete Mini protease inhibitor cocktail
(Roche). Total protein concentrations were determined and
normalized between samples. The lysates were then incubated
with prewashed NeutrAvidin coated beads at 4uC overnight. After
washes, bound surface proteins were eluted and boiled, separated
on NuPage gels (Invitrogen), and probed with rabbit anti-Cav1.2
antibody (Alomone) and mouse anti-NCX1 antibody (Abcam).
Similar expression levels of BIN1 and BIN1-BAR* were confirmed
by Western blot in the total cellular lysates. For quantitation, the
amount of surface Cav1.2 or NCX1 was normalized to input and
compared among different groups.
HeLa cells were cotransfected with human Cav1.2 along with
regulatory b2b and a2d1 subunits and BIN1-V5, harvested, and
lysed in 1% Triton X-100 Co-IP buffer (50 mM Tris pH 7.5,
150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM DTT,
1 mM NaF, 100 mM Na3VO4, 1% Triton X-100) supplemented
with Complete Mini protease inhibitor cocktail. The lysate was
then incubated with either mouse anti-V5 antibody (2 mg) or
equal amount of non-specific mouse IgG for 2 h before pulldown
with rec-protein-G-Sepharose (Invitrogen) for 1 h. Material
bound to washed beads was eluted, boiled, separated, and
probed with rabbit antibodies against Cav1.2 (Alomone) or V5
Signal Processing and Statistical Analysis
For spatial periodicity analysis in the cardiomyocytes, the
fluorescence intensity profiles were generated by ImageJ. The
frequency domain power spectrum of cardiomyocyte subsections
were generated in Matlab using FFT conversion. Next, the power
spectrum over spatial distance (1/frequency) was averaged from
five cardiomyocytes and presented in Figure 1. For T-tubule
Cav1.2 signal, intensity at each peak (corresponding to T-tubules)
was analyzed using the fluorescence intensity profiles generated by
ImageJ and Matlab. For quantitation of cell peripheral Cav1.2,
three-dimensional cross-section projection of cardiomyocytes were
generated, and fluorescence intensity within 2 mm of cell surface
was analyzed using ImageJ. In addition, a previously reported
method  using PSC Colocalization plug-in in ImageJ was used
for colocalization analysis between BIN1 and Cav1.2. For all other
statistical analysis, paired or unpaired two-tail Students t test was
performed using Prism 5 (GraphPad) software.
Figure S1 BIN1 is expressed at both T-tubules and
nuclei in cardiomyocytes Confocal images (606) of both
human (top) and mouse (bottom) cardiomyocytes. The
cells were fixed and stained with mouse anti-BIN1. DAPI was used
to label nuclei. BIN1 is localized at both nuclei and T-tubules
(scale bar: 10 mm).
Found at: doi:10.1371/journal.pbio.1000312.s001 (0.22 MB PDF)
Figure S2 Cx43 distribution is different from Cav1.2 in
cardiomyocytes. Confocal image (1006) of adult mouse
cardiomyocytes. The cells were fixed and stained with mouse
anti-Cav1.2 or rabbit anti-Cx43. Three-dimensional volume views
of Cav1.2 and Cx43 distribution are reconstructed from a stack of
1006 confocal image frames acquired at a z-step of 0.1 mm (first
column). Two-dimensional frames of Cav1.2 and Cx43 are shown
in the second column. Cardiomyocyte fluorescence intensity
profiles along 30 mm of the longitudinal axis are presented in
the third column. The bottom panel is the power spectrum over
spatial distance for Cx43 averaged from five cardiomyocytes,
which indicate that intercalated disc localized Cx43 distribution
does not have a similar pattern of Cav1.2 (see Figure 1) (scale bar:
Found at: doi:10.1371/journal.pbio.1000312.s002 (0.26 MB PDF)
Figure S3 Whole cell view of BIN1 and Cav1.2 in
cardiomyocytes. Confocal images (606) of both human (top)
and mouse (bottom) cardiomyocytes. Co-staining with mouse
antiBIN1 (green) and rabbit anti-Cav1.2 (red) indicates colocalization
of BIN1 and Cav1.2 (scale bar: 10 mm).
Found at: doi:10.1371/journal.pbio.1000312.s003 (0.25 MB PDF)
Figure S4 Cx43 does not colocalize with Cav1.2 in
cardiomyocytes. In isolated adult mouse cardiomyocytes,
costaining with Cx43 (red) and Cav1.2 (green) does not indicate
colocalization of Cx43 and Cav1.2 (scale bar: 5 mm). Pearson
colocalization coefficient and scatter plot reveal no significant
colocalization between Cx43 and Cav1.2.
Found at: doi:10.1371/journal.pbio.1000312.s004 (0.26 MB PDF)
Figure S5 Microtubule-dependent delivery of Cav1.2.
Top: Western blot indicates total cellular protein
content of Cav1.2 is not changed by nocodazole. Confocal
images (1006) of mouse cardiomyocytes subjected to control or
nocodazole treatment. Staining with rabbit anti-Cav1.2 indicates
reduction of Cav1.2 at both general cell periphery as well as along
T-tubules (scale bar: 10 mm). Quantitative data are presented in
the bottom panel (* p,0.05, Students t test).
Found at: doi:10.1371/journal.pbio.1000312.s005 (0.32 MB PDF)
Figure S6 Microtubule-dependent forward trafficking
of Cav1.2 in HL-1 cells. Surface biotinylation of endogenous
Cav1.2 in cultured HL-1 cells. Nocodazole (30 mM overnight)
reduces surface Cav1.2 expression in the presence of an
endocytosis inhibitor dynasore (80 mM). Western blot of one
representative experiment is shown in the top panel.
Quantification data of the Cav1.2 surface expression level summarized from
three separate experiments are presented in bar graph shown in
the bottom panel (** p,0.01, Students t test).
Found at: doi:10.1371/journal.pbio.1000312.s006 (0.17 MB PDF)
Figure S7 BIN1 fails to cause surface expression of
NCX1 in HL-1 cells. Surface biotinylation of endogenous
Cav1.2 and NCX1 in cultured HL-1 cells transfected with
BIN1BAR* and full-length BIN1. Western blot of one representative
experiment is shown in the left panel. Quantification of the Cav1.2
and NCX1 surface expression levels are summarized and
presented in bar graph shown in the right panel. Compared with
BIN1-BAR*, full-length BIN1 increases surface expression of
Cav1.2 but not NCX1 (** p,0.01, Students t test).
Found at: doi:10.1371/journal.pbio.1000312.s007 (0.22 MB PDF)
Figure S8 Differentiated postnatal mouse
cardiomyocytes express BIN1 and have T-tubules. (A) Confocal
images of 1-wk differentiated cardiomyocytes isolated from P3/4
postnatal mice co-stained with mouse anti-BIN1 (green) and
rabbit anti-Cav1.2 display T-tubule localization pattern. (B)
Quantitative rt-PCR data indicate postnatal mouse heart tissue
have a similar expression level of BIN1 compared to young adult
heart (8 wk).
Found at: doi:10.1371/journal.pbio.1000312.s008 (0.22 MB PDF)
Video S1 Dynamic microtubules associate with BIN1
structures. Live-cell imaging in HeLa cells transfected with
BIN1-mCherry with a-tubulin-GFP. The movie is a 2 min capture
period of images acquired at 1 s interval for a-tubulin-GFP with
400 ms exposure per frame. The a-tubulin-GFP sequence is then
merged with the BIN1-mCherry frame. Note microtubules (green)
appear to tether at BIN1 structures (red). When not interacting
with BIN1, microtubules travel rapidly.
Found at: doi:10.1371/journal.pbio.1000312.s009 (4.32 MB AVI)
We are grateful to Prof. Lily Jan for helpful discussion, Ivy Hsieh for
electron microscopy assistance, Margaret Mayes for tissue cryosectioning,
Monika Jain for technical support, Dr. Hua Wang for signal processing
assistance, and Sean Van Slyck and the California Transplant Donor
Network for human tissue.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: TTH JWS
RMS. Performed the experiments: TTH JWS DG KYC TSF. Analyzed
the data: TTH JWS JMV HMC RMS. Contributed reagents/materials/
analysis tools: BCJ HMC RMS. Wrote the paper: TTH RMS.
1. Frost A , Unger VM , De Camilli P ( 2009 ) The BAR domain superfamily: membrane-molding macromolecules . Cell 137 : 191 - 196 .
2. Butler MH , David C , Ochoa GC , Freyberg Z , Daniell L , et al. ( 1997 ) Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin/Rvs family, is concentrated in the cortical cytomatrix of axon initial segments and nodes of ranvier in brain and around T tubules in skeletal muscle . J Cell Biol 137 : 1355 - 1367 .
3. Frost A , Perera R , Roux A , Spasov K , Destaing O , et al. ( 2008 ) Structural basis of membrane invagination by F-BAR domains . Cell 132 : 807 - 817 .
4. Lee E , Marcucci M , Daniell L , Pypaert M , Weisz OA , et al. ( 2002 ) Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle . Science 297 : 1193 - 1196 .
5. Ren G , Vajjhala P , Lee JS , Winsor B , Munn AL ( 2006 ) The BAR domain proteins: molding membranes in fission, fusion, and phagy . Microbiol Mol Biol Rev 70 : 37 - 120 .
6. Muller AJ , Baker JF , DuHadaway JB , Ge K , Farmer G , et al. ( 2003 ) Targeted disruption of the murine Bin1/Amphiphysin II gene does not disable endocytosis but results in embryonic cardiomyopathy with aberrant myofibril formation . Mol Cell Biol 23 : 4295 - 4306 .
7. Chang MY , Boulden J , Katz JB , Wang L , Meyer TJ , et al. ( 2007 ) Bin1 ablation increases susceptibility to cancer during aging, particularly lung cancer . Cancer Res 67 : 7605 - 7612 .
8. Cheng H , Lederer WJ , Cannell MB ( 1993 ) Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle . Science 262 : 740 - 744 .
9. Bers DM ( 2002 ) Cardiac excitation-contraction coupling . Nature 415 : 198 - 205 .
10. Kawai M , Hussain M , Orchard CH ( 1999 ) Excitation-contraction coupling in rat ventricular myocytes after formamide-induced detubulation . Am J Physiol 277 : H603 - H609 .
11. Brette F , Salle L , Orchard CH ( 2006 ) Quantification of calcium entry at the Ttubules and surface membrane in rat ventricular myocytes . Biophys J 90 : 381 - 389 .
12. Fabiato A ( 1983 ) Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum . Am J Physiol 245 : C1 - C14 .
13. Shaw RM , Fay AJ , Puthenveedu MA , von Zastrow M , Jan YN , et al. ( 2007 ) Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions . Cell 128 : 547 - 560 .
14. Chen Y , Deng L , Maeno-Hikichi Y , Lai M , Chang S , et al. ( 2003 ) Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis . Cell 115 : 37 - 48 .
15. Itoh T , De Camilli P ( 2006 ) BAR, F-BAR (EFC) and ENTH/ANTH domains in the regulation of membrane-cytosol interfaces and membrane curvature . Biochim Biophys Acta 1761 : 897 - 912 .
16. Takenawa T , Suetsugu S ( 2007 ) The WASP-WAVE protein network: connecting the membrane to the cytoskeleton . Nat Rev Mol Cell Biol 8 : 37 - 48 .
17. Meunier B , Quaranta M , Daviet L , Hatzoglou A , Leprince C ( 2009 ) The membrane-tubulating potential of amphiphysin 2/BIN1 is dependent on the microtubule-binding cytoplasmic linker protein 170 (CLIP-170) . Eur J Cell Biol 88 : 91 - 102 .
18. Song LS , Sobie EA , McCulle S , Lederer WJ , Balke CW , et al. ( 2006 ) Orphaned ryanodine receptors in the failing heart . Proc Natl Acad Sci U S A 103 : 4305 - 4310 .
19. Soeller C , Cannell MB ( 1999 ) Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques . Circ Res 84 : 266 - 275 .
20. Chu PJ , Rivera JF , Arnold DB ( 2006 ) A role for Kif17 in transport of Kv4 .2. J Biol Chem 281 : 365 - 373 .
21. Nejsum LN , Nelson WJ ( 2007 ) A molecular mechanism directly linking Ecadherin adhesion to initiation of epithelial cell surface polarity . J Cell Biol 178 : 323 - 335 .
22. Macia E , Ehrlich M , Massol R , Boucrot E , Brunner C , et al. ( 2006 ) Dynasore, a cell-permeable inhibitor of dynamin . Dev Cell 10 : 839 - 850 .
23. White SM , Constantin PE , Claycomb WC ( 2004 ) Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function . Am J Physiol Heart Circ Physiol 286 : H823 - H829 .
24. Nicot AS , Toussaint A , Tosch V , Kretz C , Wallgren-Pettersson C , et al. ( 2007 ) Mutations in amphiphysin 2 (BIN1) disrupt interaction with dynamin 2 and cause autosomal recessive centronuclear myopathy . Nat Genet 39 : 1134 - 1139 .
25. Seki S , Nagashima M , Yamada Y , Tsutsuura M , Kobayashi T , et al. ( 2003 ) Fetal and postnatal development of Ca2+ transients and Ca2+ sparks in rat cardiomyocytes . Cardiovasc Res 58 : 535 - 548 .
26. Snopko RM , Ramos-Franco J , Di Maio A , Karko KL , Manley C , et al. ( 2008 ) Ca2+ sparks and cellular distribution of ryanodine receptors in developing cardiomyocytes from rat . J Mol Cell Cardiol 44 : 1032 - 1044 .
27. Perissel B , Charbonne F , Moalic JM , Malet P ( 1980 ) Initial stages of trypsinized cell culture of cardiac myoblasts: ultrastructural data . J Mol Cell Cardiol 12 : 63 - 75 .
28. Osinska HE , Lemanski LF ( 1993 ) Immunofluorescent studies on Z-lineassociated protein in cultured cardiomyocytes from neonatal hamsters . Cell Tissue Res 271 : 59 - 67 .
29. Mohler PJ , Yoon W , Bennett V ( 2004 ) Ankyrin-B targets beta2-spectrin to an intracellular compartment in neonatal cardiomyocytes . J Biol Chem 279 : 40185 - 40193 .
30. Mohler PJ , Gramolini AO , Bennett V ( 2002 ) The ankyrin-B C-terminal domain determines activity of ankyrin-B/G chimeras in rescue of abnormal inositol 1,4,5-trisphosphate and ryanodine receptor distribution in ankyrin-B (2/2) neonatal cardiomyocytes . J Biol Chem 277 : 10599 - 10607 .
31. Kim KH , Kim TG , Micales BK , Lyons GE , Lee Y ( 2007 ) Dynamic expression patterns of leucine-rich repeat containing protein 10 in the heart . Dev Dyn 236 : 2225 - 2234 .
32. Jung M , Poepping I , Perrot A , Ellmer AE , Wienker TF , et al. ( 1999 ) Investigation of a family with autosomal dominant dilated cardiomyopathy defines a novel locus on chromosome 2q14-q22 . Am J Hum Genet 65 : 1068 - 1077 .
33. Chien AJ , Zhao X , Shirokov RE , Puri TS , Chang CF , et al. ( 1995 ) Roles of a membrane-localized beta subunit in the formation and targeting of functional Ltype Ca2+ channels . J Biol Chem 270 : 30036 - 30044 .
34. Gwathmey JK , Copelas L , MacKinnon R , Schoen FJ , Feldman MD , et al. ( 1987 ) Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure . Circ Res 61 : 70 - 76 .
35. Beuckelmann DJ , Nabauer M , Erdmann E ( 1992 ) Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure . Circulation 85 : 1046 - 1055 .
36. Sipido KR , Stankovicova T , Flameng W , Vanhaecke J , Verdonck F ( 1998 ) Frequency dependence of Ca2+ release from the sarcoplasmic reticulum in human ventricular myocytes from end-stage heart failure . Cardiovasc Res 37 : 478 - 488 .
37. Harding SE , Davies CH , Wynne DG , Poole-Wilson PA ( 1994 ) Contractile function and response to agonists in myocytes from failing human heart . Eur Heart J 15 Suppl D : 35 - 36 .
38. Hasenfuss G ( 1998 ) Alterations of calcium-regulatory proteins in heart failure . Cardiovasc Res 37 : 279 - 289 .
39. Hasenfuss G , Schillinger W , Lehnart SE , Preuss M , Pieske B , et al. ( 1999 ) Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium . Circulation 99 : 641 - 648 .
40. Lehnart SE , Wehrens XH , Reiken S , Warrier S , Belevych AE , et al. ( 2005 ) Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias . Cell 123 : 25 - 35 .
41. Marx SO , Reiken S , Hisamatsu Y , Jayaraman T , Burkhoff D , et al. ( 2000 ) PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts . Cell 101 : 365 - 376 .
42. Gomez AM , Valdivia HH , Cheng H , Lederer MR , Santana LF , et al. ( 1997 ) Defective excitation-contraction coupling in experimental cardiac hypertrophy and heart failure . Science 276 : 800 - 806 .
43. Litwin SE , Zhang D , Bridge JH ( 2000 ) Dyssynchronous Ca(2+) sparks in myocytes from infarcted hearts . Circ Res 87 : 1040 - 1047 .
44. Chen X , Piacentino V III , Furukawa S , Goldman B , Margulies KB , et al. ( 2002 ) L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices . Circ Res 91 : 517 - 524 .
45. He J , Conklin MW , Foell JD , Wolff MR , Haworth RA , et al. ( 2001 ) Reduction in density of transverse tubules and L-type Ca(2+) channels in canine tachycardia-induced heart failure . Cardiovasc Res 49 : 298 - 307 .
46. Takahashi SX , Miriyala J , Colecraft HM ( 2004 ) Membrane-associated guanylate kinase-like properties of beta-subunits required for modulation of voltage-dependent Ca2+ channels . Proc Natl Acad Sci U S A 101 : 7193 - 7198 .
47. Pease DC ( 1964 ) Histology techniques for electron microscopy . New York and London : Academic Press.
48. Stenberg PE , Shuman MA , Levine SP , Bainton DF ( 1984 ) Redistribution of alpha-granules and their contents in thrombin-stimulated platelets . J Cell Biol 98 : 748 - 760 .
49. McCaffery JM , Farquhar MG ( 1995 ) Localization of GTPases by indirect immunofluorescence and immunoelectron microscopy . Methods Enzymol 257 : 259 - 279 .
50. Peters PJ , Bos E , Griekspoor A ( 2006 ) Cryo-immunogold electron microscopy . Curr Protoc Cell Biol Chapter 4: Unit 4 7.
51. Dipla K , Mattiello JA , Jeevanandam V , Houser SR , Margulies KB ( 1998 ) Myocyte recovery after mechanical circulatory support in humans with endstage heart failure . Circulation 97 : 2316 - 2322 .
52. Beuckelmann DJ , Nabauer M , Erdmann E ( 1991 ) Characteristics of calciumcurrent in isolated human ventricular myocytes from patients with terminal heart failure . J Mol Cell Cardiol 23 : 929 - 937 .
53. O'Connell TD , Rodrigo MC , Simpson PC ( 2007 ) Isolation and culture of adult mouse cardiac myocytes . Methods Mol Biol 357 : 271 - 296 .
54. French AP , Mills S , Swarup R , Bennett MJ , Pridmore TP ( 2008 ) Colocalization of fluorescent markers in confocal microscope images of plant cells . Nat Protoc 3 : 619 - 628 .