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Journal of Molecular and Cellular Cardiology
journal homepage: www.elsevier.com/locate/yjmcc
Location and function of transient receptor potential canonical channel 1 in
ventricular myocytes
Qinghua Hu
a,c,1
, Azmi A. Ahmad
a,b,1
, Thomas Seidel
a
, Chris Hunter
a
, Molly Streiff
a,b
,
Linda Nikolova
d
, Kenneth W. Spitzer
a
, Frank B. Sachse
a,b,⁎
a
Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT 84112, USA
b
Department of Biomedical Engineering, University of Utah, Salt Lake City, UT 84112, USA
c
Department of Cardiovascular Surgery, Xiangya Hospital, Central-South University, Changsha, Hunan 410078, China
d
Core Research Facilities, Health Sciences Center, University of Utah, Salt Lake City, UT 84112, USA
ARTICLE INFO
Keywords:
TRPC1 channels
Ventricular myocyte
Sarcoplasmic reticulum
Calcium signaling
ABSTRACT
Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While
TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is
poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca
2+
signaling in
mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and
quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ven-
tricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular
proteins including sarco/endoplasmic reticulum Ca
2+
ATPase 2 in the sarcoplasmic reticulum (SR).
Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma in-
cluding the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the
intracellular location of TRPC1. We investigated Ca
2+
signaling in NRVMs after adenoviral TRPC1 over-
expression or silencing. In NRVMs bathed in Na
+
and Ca
2+
free solution, TRPC1 overexpression and silencing
was associated with a decreased and increased SR Ca
2+
content, respectively. In isolated rabbit cardiomyocytes
bathed in Na
+
and Ca
2+
free solution, we found an increased decay of the cytosolic Ca
2+
concentration [Ca
2+
]
i
and increased SR Ca
2+
content in the presence of the TRPC channel blocker SKF-96365. In a computational
model of rabbit ventricular myocytes at physiological pacing rates, Ca
2+
leak through SR TRPC channels in-
creased the systolic and diastolic [Ca
2+
]
i
with only minor effects on the action potential and SR Ca
2+
content.
Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ven-
tricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca
2+
leak in car-
diomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will
guide us to an understanding of TRPC1 channels as modulators of [Ca
2+
]
i
and contractility in cardiomyocytes.
1. Introduction
Transient receptor potential (TRP) channels were originally dis-
covered in Drosophila [1]. Subsequently, a variety of TRP-related
channels were identified in mammals. Based on homology of protein
sequences TRP-related channels have been classified into seven families
[2], including the family of transient receptor potential canonical
(TRPC) channels that are expressed in the mammalian heart. While
recent evidence suggests that TRPC channels are critical effectors in
cardiac hypertrophy and heart failure [3–7], their physiological role in
cardiac cells is still not understood [8]. Suggested roles for TRPC
channels in these cells include store-operated and receptor-operated
Ca
2+
entry [9]. Store-operated Ca
2+
entry is a mechanism by which the
Ca
2+
-depleted sarcoplasmic reticulum (SR) is refilled by Ca
2+
flux
through sarcolemmal ion channels into the cytosol and subsequent
uptake in the SR. Receptor-operated Ca
2+
entry is a mechanism by
https://doi.org/10.1016/j.yjmcc.2020.01.008
Received 17 August 2019; Received in revised form 16 December 2019; Accepted 21 January 2020
Abbreviations: eGFP, enhanced green fluorescent protein; NRVMs, neonatal rat ventricular myocytes; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; sh, short
hairpin; TRP, transient receptor potential; TRPC1, transient receptor potential canonical 1
⁎
Corresponding author at: University of Utah, Nora Eccles Harrison Cardiovascular Research and Training Institute, 95 South 2000 East, Salt Lake City, UT 84112-
5000, USA.
E-mail address: frank.sachse@utah.edu (F.B. Sachse).
1
Contributed equally to this paper
Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
Available online 23 January 2020
0022-2828/ © 2020 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
T
which Ca
2+
flux through sarcolemmal ion channels is controlled by a
receptor, commonly a G-protein-coupled receptor. In addition to a role
in store-operated and receptor-operated Ca
2+
entry TRPC channels in
cardiomyocytes have been linked to mechano-electrical feedback, i.e.
modulation of cellular electrophysiology by mechanical stimuli. This
suggested role is based on studies indicating that some TRPC channels,
including TRPC1 channels, are stretch-activated [10,11].
A major obstacle to identification of a functional role of TRPC
channels in cardiomyocytes is the diversity of information concerning
subcellular location of the channels [8]. The location of TRPC channels
in ventricular cardiomyocytes was primarily investigated in rodents.
Several sarcolemmal locations were proposed: outer sarcolemma
[12–14], transverse tubular system (t-system) [12–18], and sarcolemma
at the intercalated disks [15]. Two types of sub-sarcolemmal location
were suggested: subsarcolemmal vesicles and peripheral region of the
myocytes [19,20]. While a location of TRPC channels in intracellular
membranes has been indicated in some studies [21,22], the specific
organelle was in general not specified. Interestingly, a study on skeletal
muscle in rodent reported that TRPC1 resides in the SR [23]. This study
suggested a functional role of TRPC1 channels in SR Ca
2+
leak in
skeletal myocytes.
Here, we shed light on the functional role of TRPC channels in
ventricular cardiomyocytes. We focus on TRPC1, which is abundantly
expressed in these cells. We tested the hypothesis that TRPC1 channels
localized in the SR membrane of ventricular cardiomyocytes contribute
to SR Ca
2+
leak. In these cells, SR Ca
2+
leak is commonly explained by
ryanodine receptors (RyRs) [24].
First, we investigated the spatial distribution of TRPC1 in neonatal
rat ventricular myocytes (NRVMs). NRVMs allowed us to overexpress
and silence TRPC1 expression using adenoviral TRPC1-eGFP and short
hairpin (sh)RNA TRPC1-eGFP constructs, respectively. Importantly,
NRVMs do not exhibit a t-system, which simplifies establishing a spatial
relationship of TRPC1 to the sarcolemma. Next, we studied adult rabbit
ventricular myocytes to relate findings from cultured neonatal cells to
adult native cells with a t-system similar as in human ventricular
myocytes. We performed three-dimensional (3D) confocal microscopy
to visualize and reconstruct the distribution of TRPC1, sarcolemma, SR,
and cytoskeletal proteins. We measured colocalization to investigate
the spatial relationship of TRPC1 channels with the sarcolemma and
various proteins. In addition, we applied super-resolution and im-
munoelectron microscopy to study the distribution of TRPC1 at nan-
ometer scale. Subsequently, we used a cytosolic Ca
2+
indicator and
fluorescence microscopy in NRVMs to investigate if TRPC1 over-
expression and silencing modulate SR Ca
2+
leak. These studies were
accompanied by studies on NRVMs and adult rabbit ventricular myo-
cytes using a TRPC channel blocker. Finally, we applied a computa-
tional model of rabbit ventricular myocytes to explore a potential
functional role of TRPC1 channels in electrophysiology and in-
tracellular Ca
2+
signaling.
2. Materials and methods
All studies were conducted in accordance with National Institutes of
Health Guidelines for the Care and Use of Animals and reviewed by the
Institutional Animal Care and Use Committee at the University of Utah,
where the work was performed. An additional Methods section is
available in Supplementary Material.
2.1. Preparation, adenoviral infection and culture of NRVMs
Cells were enzymatically isolated from 1-day old rats (NCIS,
Worthington Biochemical Corporation, Lakewood, NJ, USA). NRVMs
were subsequently separated from fibroblasts and plated at 175-200 k
density in 24 well tissue culture plates containing 5 mm coverslips
treated with fibronectin. In some studies, NRVMs were then infected
with an adenoviral vector containing a human TRPC1 attached to
enhanced green fluorescent protein (TRPC1-eGFP) at 200 multi-
plication of infection (MOI) or eGFP (Cat No. 1060) as control at 25
MOI. In some studies, we also applied an adenoviral vector with human
TRPC1 attached to enhanced green fluorescent protein and 6 HIS fused
to the N-terminal. Furthermore, we infected NRVMs with shRNA TRPC1
with eGFP marker (shRNA-TRPC1-eGFP, Cat No. shADV-226,536) to
silence TRPC1 expression, or a scrambled RNA with eGFP marker
(scRNA-eGFP, Cat No. 1122) as shRNA-TRPC1 control. Both infections
were performed at 80 MOI. All viral vectors had a backbone of type 5
(dE1/E3), and were produced by Vector Biolabs (Malvern, PA, USA). At
24 h after infection, cells were washed with DMEM to remove the virus.
Infected cells were then maintained and cultured for a total of 4–5 days
by regularly exchanging the culture media, and incubating them in a
humidity and CO
2
controlled incubator at 37 °C.
2.2. Immunolabeling of myocytes
NRVMs plated on coverslips were fixed for 15 min with 1% paraf-
ormaldehyde. Cells were permeabilized using 0.3% Triton X-100 (VWR
International, Radnor, PA, USA) for 18 min and then bathed in image-iT
FX Signal Enhancer (Thermo Fisher Scientific, Waltham, MA, USA) for
30 min and in 10% normal donkey serum (Millipore, Billerica, MA,
USA) for 60 min. Cells were then incubated with primary antibodies for
TRPC1 (T8666-09A, Supplementary Methods) and sarco/endoplasmic
reticulum Ca
2+
ATPase 2 (SERCA2) (MA3–910, Thermo Fisher
Scientific) overnight at 4 °C. Subsequently, cells were incubated with
secondary antibodies, a donkey anti-goat conjugated to Alexa Fluor 633
(Invitrogen, Carlsbad, California, USA) and a donkey anti-mouse con-
jugated to Alexa Fluor 555 for 60 min at room temperature. Finally,
cells were incubated with DAPI (D3571, Thermo Fisher Scientific) for
15 min to stain nuclei. Cells were triple-rinsed with phosphate-buffered
saline (PBS) for 15 min between each step.
Rabbit cardiomyocytes were isolated using enzymatic digestion
(Supplementary Methods). Cell pellets were incubated with wheat germ
agglutinin (WGA) conjugated to Alexa Fluor-633 for 20 min, followed
by 15 min of fixation with 2% paraformaldehyde. Before applying
primary antibodies, the cells were permeabilized using 0.1% Triton X-
100 and blocked using 10% normal donkey serum. Commonly, two
different primary antibodies were applied simultaneously, one raised in
goat for labeling TRPC1 channels and the other raised in mouse for
labeling other proteins. The cells were incubated with the primary
antibodies overnight at 4 °C. Two secondary antibodies, a donkey anti-
goat antibody conjugated to Alexa Fluor 488 and a donkey anti-mouse
antibody conjugated to Alexa Fluor 555, were then applied for 2 h at
room temperature.
We assessed specificity of the TRPC1 antibody using confocal mi-
croscopy of skeletal muscle tissues from WT and TRPC1 knockout mice
(Supplementary Methods and Fig. S1). Furthermore, we assessed our
TRPC1 labeling protocol in rabbit cardiomyocytes by omission of the
primary antibody and pre-incubation of the primary antibody with a
blocking peptide, which both eliminated the TRPC1 signal (Fig. S2).
2.3. Confocal imaging and image processing
For acquiring 3D stacks of NRVMs, we used a Leica SP8 TCS mi-
croscope (Leica Microsystems, Wetzlar, Germany) equipped with
GaAsP-HyD detectors and a 40× oil immersion lens (numerical aper-
ture: 1.2). Alexa Fluor 488 was excited with a laser wavelength of
488 nm and emitted light was acquired after band-pass filtering from
491 to 555 nm. Alexa Fluor 555 and 633 were excited at 561 and
633 nm wavelengths, respectively. Fluorescence for excitation at 561
and 633 nm was collected at 566–604 and 638–775 nm, respectively.
DAPI signal was excited with a 405 nm laser and collected at
410–570 nm.
For 3D imaging of segments of rabbit myocytes, we used a Zeiss LSM
5 Duo microscope (Carl Zeiss, Jena, Germany) equipped with a 63× oil
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
114
immersion lens (numerical aperture: 1.4). Alexa Fluor 488 was excited
with a 488 nm laser and emitted light was acquired after long pass
filtering at 505 nm. Alexa Fluor 555 and Alexa Fluor 633 were excited
at a wavelength of 543 nm and 633 nm, respectively. A 560 nm and
650 nm long pass filter was applied for light emitted from Alexa Fluor
555 and Alexa Fluor 633, respectively.
We acquired image stacks with a voxel size of 0.1 × 0.1 × 0.1 μm.
The image stacks were preprocessed including noise reduction, decon-
volution, background correction and attenuation correction as de-
scribed previously [25]. We applied Pearson's correlation coefficient R
r
to assess colocalization in the images (Supplementary Methods).
2.4. Measurement of [Ca
2+
]
i
in myocytes
Experiments were conducted on NRVMs at 4–5 days post-infection.
[Ca
2+
]
i
was measured with a Leica SP8 TCS confocal microscope
equipped with a 40× oil immersion lens (numerical aperture: 1.2) and
GaAsP-HyD detectors. NRVMs were loaded with 10 μM Rhod-3 AM
(R10145, Thermo-Fisher Scientific) in modified Tyrode solution at
37 °C for 45 min. After transfer into an imaging chamber, cells were
superfused with the modified Tyrode solution at room temperature
(22 ± 1 °C). A 488 nm laser was applied to excite eGFP, and emitted
light was collected after a 491–555 nm bandpass filter. A 561 nm laser
was then used to excite Rhod-3 Ca
2+
dye, and emitted light was col-
lected using a 566 nm long pass filter. Na
+
and Ca
2+
free Tyrode-like
solution was used to block the sodium‑calcium exchanger and other
sarcolemmal Ca
2+
currents.
Image sequences were acquired at a rate of 28 ms/image. Cells were
initially paced by field stimulation at 0.5 Hz in modified Tyrode solu-
tion until reaching steady state Ca
2+
signals. We then turned off the
stimulation and rapidly switched to Na
+
and Ca
2+
free solution for
2 min. Afterwards we rapidly applied 20 mM caffeine in the Na
+
and
Ca
2+
free solution to cause SR Ca
2+
release and to estimate SR Ca
2+
content as described previously [26]. The protocol was applied to
NRVMs infected with eGFP, TRPC1-eGFP, shRNA-TRPC1-eGFP, and
scRNA-eGFP. [Ca
2+
]
i
transients were extracted from Rhod3 signal by
cropping cytosolic regions with homogeneous intensities as described in
Fig. S3. For analyses of SR Ca
2+
content measurements, traces with
multiple transients during the Na
+
and Ca
2+
free period were ex-
cluded.
Similar studies on eGFP infected NRVMs were performed to assess
RyR Ca
2+
leak. SR Ca
2+
content and leak were assessed with or without
application of 1 mM tetracaine (Sigma-Aldrich) during the application
of Na
+
and Ca
2+
free solution. Furthermore, we carried out studies
using the TRPC1-eGFP and eGFP constructs with or without application
of 5 μM SKF-96365 (Sigma-Aldrich), a general TRPC channel blocker.
SKF-96365 was dissolved in DMSO before adding it to Na
+
and Ca
2+
free solution. Finally, we measured amplitudes of [Ca
2+
]
i
transients
from the various experiments using eGFP, TRPC1-eGFP, and shRNA-
TRPC1-eGFP infected NRVMs.
Our protocol for measurement of [Ca
2+
]
i
in rabbit ventricular
myocytes is described in supplemental material. We applied SKF-96365
at a concentration of 5 μM as a non-specific blocker of TRPC channels
[13].
2.5. Modeling of SR Ca
2+
leak and effects on [Ca
2+
]
i
Mathematical models of NRVMs and adult rabbit ventricular myo-
cytes were used to shed light on effects of SR Ca
2+
leak through TRPC1
channels on cellular electrophysiology and Ca
2+
signaling for physio-
logical pacing rates. We refer to the supplement for an introduction to
our NRVM model. Our model of adult rabbit ventricular myocytes was
based on a previously developed model [27], which comprises a de-
scription of passive SR Ca
2+
leak into the junctional space through RyR
channels. We added a description of Ca
2+
fluxes through TRPC1
channels J
SR,TRPC
from the SR into the cytosol:
=+ +
J K ([Ca ] [Ca ] )
SR,TRPC SR,TRPC 2SR 2i
with the rate of leak K
SR,TRPC
and the SR Ca
2+
concentration [Ca
2+
]
SR
.
The rate K
SR,TRPC
was determined by fitting of self-ratioed cytosolic
Ca
2+
signals measured in the presence and absence of SKF-96365 to
self-ratioed [Ca
2+
]
i
calculated with the myocyte model. Simulations
with the model were carried out using JSim (version 2.13) [28]. Si-
mulation results after 1 min of pacing at 2, 3 and 4 Hz were analyzed
for reduced, normal and increased Ca
2+
fluxes through TRPC1 chan-
nels.
2.6. Statistical analysis
Data are presented as mean ± standard error. Statistical analyses
were performed in Matlab version R2012b and higher (Mathworks Inc.,
Natick, MA, USA). Comparison of experimental data was performed
using a one-way analysis of variables (ANOVA). Comparison of data
from epifluorescence microscopy was based on the paired student t-test.
Differences were considered significant for P-values less than 0.01 or
0.05 where noted.
3. Results
3.1. Spatial distribution of TRPC1 in NRVMs
Applying confocal microscopy, we investigated the spatial dis-
tribution of wild-type (WT) TRPC1 in NRVMs that were cultured for
4–6 days, then fixed and labeled. Example sections from unprocessed
and deconvolved 3D image stacks are presented in Fig. S4 and 1, re-
spectively. Remarkably, TRPC1 exhibited a striated intracellular dis-
tribution (Fig. 1A). SERCA2 labeling presented a similar striated pat-
tern, accompanied by an irregular network pattern spanning
throughout the cell (Fig. 1B). The spatial distribution is particularly
apparent in enlarged images (Fig. 1D and E). SERCA2 and TRPC1 ap-
pear to some degree colocalized in the overlay image (Fig. 1G).
After infection with TRPC1-eGFP constructs and culture of 4–5 days,
NRVMs showed similar TRPC1 and SERCA2 patterns (Fig. 2A-D) as WT
NRVMs (Fig. 1). Unprocessed stacks are shown in Fig. S5. Fluorescence
of eGFP confirmed adenoviral TRPC1 expression (Fig. 2E and F).
Overlay images present a similar colocalization of TRPC1 and SERCA2
antibody associated fluorescence (Fig. 2G) as in WT NRVMs, but only
weak colocalization of TRPC1 and TRPC1-eGFP fluorescence (Fig. 2H)
as well as partial colocalization of TRPC1-eGFP with SERCA2 fluores-
cence (Fig. 2I).
WT NRVMs exhibited high colocalization of SERCA2 and TRPC1
(R
r
= 0.4 ± 0.03, n
cells
= 5). Similarly, R
r
values for TRPC1 coloca-
lization with SERCA2 across eGFP, TRPC1-eGFP, and shRNA-TRPC1-
eGFP infected NRVMs were 0.46 ± 0.03 (n
cells
= 5), 0.41 ± 0.03
(n
cells
= 5), and 0.47 ± 0.03 (n
cells
= 5), respectively (Fig. 2J). Dif-
ferences were not significant between the groups. Colocalization of
eGFP signal with SERCA2 was higher for TRPC1-eGFP versus eGFP and
shRNA-TRPC1-eGFP infected cells (0.41 ± 0.04 versus 0.26 ± 0.02
and 0.22 ± 0.04, respectively P< .05, n
cells
= 5) (Fig. 2K).
Example images from confocal microscopy of TRPC1 labeled
NRVMs infected with eGFP (7 images, ~30 cells), TRPC1-eGFP (14
images, ~40 cells), shRNA-TRPC1-eGFP (10 images, ~25 cells) and
scrambled RNA constructs (3 images, ~30 cells) are shown in Fig. S6. In
shRNA-TRPC1-eGFP infected cells, TRPC1 signals were small and
striations rare (Fig. S6C) suggesting silencing of TRPC1 protein ex-
pression.
We acquired super-resolution images of TRPC1 and SERCA2 in WT
NRVMs using single molecule localization (Supplementary Methods).
The NRVMs exhibited a striated arrangement of TRPC1 clusters of
variable size (Fig. 3A). SERCA2 exhibited a similar pattern and was in
part in close proximity to TRPC1 (Fig. 3B and C).
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
115
3.2. Spatial relationship of TRPC1, SERCA2 and cytoskeletal proteins in
rabbit ventricular myocytes
We imaged rabbit ventricular myocytes labeled with WGA and
TRPC1 antibodies. WGA served as a marker of the outer sarcolemma
and t-system. Exemplary images from co-labeling of WGA and TRPC1
are presented in Fig. 4. TRPC1 exhibited a striated transversal dis-
tribution along Z-lines as indicated by the t-system. The striated dis-
tribution is particularly evident in 3D visualization of cell segments
(Fig. 4E). The majority of signal for TRPC1 did not overlap with WGA
signal, suggesting that TRPC1 is localized in an intracellular membrane.
We confirmed presence of TRPC1 in rabbit ventricular myocardium
using RNA sequencing (Supplementary Material, Fig. S7 and [29]).
TRPC1 mRNA expression was higher than expression of other TRPCs.
Similar as for NRVMs, we investigated whether TRPC1 is located in
the SR of adult rabbit cardiomyocytes, using SERCA2 as a marker
(Fig. 5). SERCA2 labeling presented a lattice-like pattern including
transverse components adjacent to the Z-lines and longitudinal com-
ponents spanning throughout the whole cell (Fig. 5B), which is in
agreement with previous studies [30]. Visual inspection of the overlay
images indicates that a large portion of TRPC1 was colocalized with
transverse components of SERCA2 (Fig. 5C and D).
It has been suggested that the cytoskeleton is a major modulator of
localization and function of the TRPC channels [31,32]. Thus, we
characterized the spatial relationship between TRPC1 and cytoskeletal
proteins using confocal microscopy (Fig. 5E-H). Major components of
the cytoskeleton are actin filaments, microtubules, and intermediate
filaments. We therefore investigated α-actinin, which is an established
marker for the Z-lines of sarcomeres, and vinculin, which is abundantly
expressed in the costamere and intercalated disks and plays a role in
anchoring actin filaments to integrins. We also investigated β-tubulin,
one of the subunits forming the heterodimer tubulin of microtubules,
Fig. 1. Confocal microscopic images of fixed WT NRVMs labeled for TRPC1, SERCA2 and nuclei. The images were extracted from deconvolved 3D image stacks. (A)
The spatial distribution of TRPC1 exhibits a striated pattern. (B) SERCA2, a marker for SR, presents a striated pattern accompanied with mesh-network pattern. (C)
DAPI was used a marker for the nucleus. (D-F) Enlarged regions marked with box in (A-C), respectively. (G) Enlarged overlay of (D-F). Orange indicates overlap of
TRPC1 and SERCA2 associated fluorescence. The scale bar in (A) applies to (B) and (C). Scale bar in (D) applies to (E) and (F).
Fig. 2. Confocal microscopic images of fixed TRPC1-eGFP infected NRVMs and colocalization analyses. (A) Similar as in WT NRVMS, antibody labeling reveals a
striated pattern of TRPC1. (B) Enlarged region marked with box in (A). (C) SERCA2 antibody indicating SR. (D) Enlarged region marked with box in (C). (E) Image of
expressed TRPC1-eGFP construct. (F) Enlarged region marked with box in (E). (G) Overlay of (B) and (D). (H) Overlay of (B) and (F). (I) Overlay of (D) and (F). (J)
Pearson correlation coefficient (R
r
), calculated from image stacks of TRPC1 and SERCA antibody in different infection groups. (K) R
r
calculated from image stacks of
eGFP signal and SERCA antibody associated fluorescence in three infection groups. The scale bar in (A) applies to (C) and (E). Scale bar in (B) applies to (D), (F) and
(G-I). Brackets mark significant differences (P< .05).
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
116
and desmin, a type III intermediate filament located adjacent to Z-lines.
TRPC1 was highly colocalized with α-actinin (Fig. 5E) and desmin
(Fig. 5F). Beta-tubulin indicated a predominantly longitudinal or-
ientation of microtubules, and its overlap with TRPC1 signals was small
(Fig. 5G). TRPC1 was colocalized with vinculin (Fig. 5H) to a similar
degree as with SERCA2.
In addition to visual inspection, we assessed the colocalization of
TRPC1, WGA, SERCA2A and proteins of the cytoskeleton by calculating
Pearson's correlation coefficient R
r
from 3D image stacks. Our results,
using image stacks from cell segments (n
cells
= 48), showed high values
of R
r
for TRPC1 with sarcomeric α-actinin (0.647 ± 0.033), indicating
spatial adjacency (Fig. 5I). Values of R
r
were moderate for TRPC1 with
desmin, vinculin and SERCA2. Small values of R
r
for TRPC1 with β-
tubulin and WGA indicate remoteness of TRPC1 from microtubules and
sarcolemma, respectively. The colocalization of WGA with TRPC1 was
as small as with α-actinin, β-tubulin and desmin (Fig. 5J).
3.3. Immunoelectron microscopy of TRPC1 in rabbit ventricular myocytes
Using immunoelectron microscopy we imaged the distribution of
TRPC1 at nanometer resolution (Supplementary Material, Fig. S8).
TRPC1 was extensively marked adjacent to the Z-lines and along the
sarcomeres, but only few markers were found elsewhere. The abun-
dance of the TRPC1 labeling appeared higher in regions close (within
200 nm) to the Z-lines than elsewhere, which agreed with our findings
from confocal microscopy. However, instead of the regular striated
pattern observed in confocal microscopy, a clustered distribution of
TRPC1 was found in electron microscopy. In our images from
Fig. 3. Example super-resolution images of fixed WT NRVMs labeled with (A) TRPC1 and (B) SERCA2 antibody. (C) Overlay of images (A) and (B). (D-F) Enlarged
regions marked by box in (A-C). TRPC1 and SERCA2 present a striated clustered arrangement. The scale bar in (A) applies to (B) and (C). Scale bar in (D) applies to
(E) and (F).
Fig. 4. Confocal microscopic images of TRPC1 and sarcolemma in adult rabbit ventricular myocyte. (A) TRPC1 exhibits a transversely striated distribution with the
majority of TRPC1 within the cell. (B) WGA was used a marker for sarcolemma including t-system. (C) Overlay of (A) and (B). (D) Enlarged region marked with box in
(C). (E) Three-dimensional visualization of cell segment.
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
117
immunoelectron microscopy TRPC1 was not visible in the t-system (Fig.
S8A) and outer sarcolemma (Fig. S8B), supporting the hypothesis that
TRPC1 is located within the myocyte. Omission of primary antibodies
served as negative control (Fig. S9).
3.4. Measurement of SR Ca
2+
content in NRVMs
Our studies on TRPC1 location in NRVMs suggested that the chan-
nels are not in the sarcolemma, but in an organelle. Using confocal
imaging and a Ca
2+
sensitive dye we investigated SR Ca
2+
content in
NRVMs infected with eGFP, TRPC1-eGFP and shRNA-TRPC1-eGFP
constructs (Fig. 6A, B and C, respectively). The protocol involved pa-
cing of NRVMs, followed by bathing of quiescent cells in Na
+
and Ca
2+
free solution for 2 min, followed by rapid application of caffeine (Fig.
S3). Rapid application of caffeine led to a sudden increase of the Ca
2+
signal, which is explained by RyR channel opening and Ca
2+
release
from the SR into the cytosol. We evaluated the SR content by measuring
the amplitude of caffeine-induced peaks (Fig. 7). The SR Ca
2+
content
was measured in NRVMs infected with either eGFP (Fig. 7A), TRPC1-
eGFP (Fig. 7B) or shRNA-TRPC1-eGFP (Fig. 7C). SR Ca
2+
content was
Fig. 5. Spatial distribution of TRPC1 in rabbit ventricular myocytes. Confocal microscopic images of (A) TRPC1 and (B) SERCA2. (C) Overlay of (A) and (B). (D)
Enlarged region marked with box in (C). Yellow regions indicate colocalization of SERCA2 and TRPC1. The scale bar in (A) applies to (B) and (C). Confocal
microscopic images of TRPC1 with (E) α-actinin, (F) desmin, (G) β-tubulin and (H) vinculin. (I) Pearson correlation coefficients (R
r
) calculated from image stacks of
TRPC1, intracellular proteins and WGA. (J) R
r
determined from image stacks of WGA with TRPC1 and intracellular proteins. Brackets mark significant differences
(P< .01). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Confocal microscopic images of living NRVMs infected with (A) eGFP at 25 MOI (B) TRPC1-eGFP at 200 MOI, and (C) shRNA-TRPC1-eGFP at 80 MOI.
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
118
smaller in NRVMs overexpressing TRPC1 (1.64 ± 0.21, n
cells
= 21)
than in NRVMs infected with eGFP (3.18 ± 0.23, n
cells
= 15,
P< .01). In contrast, SR Ca
2+
content was higher in NRVMs expres-
sing shRNA-TRPC1 (4.30 ± 0.49, n
cells
= 12, P< .05) than in
NRVMs expressing TRPC1-eGFP or eGFP. Studies were done on at least
8 litters per group.
Next, we studied effects of the TRPC channel inhibitor SKF-96365
on infected NRVMs (Fig. S10). Application of SKF-96365 increased SR
Ca
2+
content in both TRPC1-eGFP (2.56 ± 0.45, n
cells
= 11, P < .05)
and eGFP (2.92 ± 0.43, n
cells
= 13, P < .01) versus TRPC1-eGFP
infected cells in the absence of SKF-96365. As in Fig. 7D, NRVMs
overexpressing TRPC1-eGFP had reduced SR Ca
2+
content compared to
eGFP NRVMs (1.56 ± 0.22, n
cells
= 17 vs. 2.5 ± 0.24, n
cells
= 14,
P< .01). Studies were done on at least 5 litters per group.
To study possible effects of short-hairpin infection on NRVMs, we
conducted similar measurements of SR Ca
2+
content with a scrambled
RNA vector to serve as a separate control for the shRNA-TRPC1-eGFP
group (Fig. S11). The difference was not significant between eGFP and
scRNA-eGFP cells, suggesting that effects of shRNA-TRPC1-eGFP are
due to silencing of TRPC1 expression.
To assess RyR Ca
2+
leak, we applied 1 mM tetracaine. Example
traces with and without tetracaine application are shown in Fig. S12A
and B, respectively. We found differences in SR Ca
2+
content (Fig.
S12C) and self-ratioed [Ca
2+
]
i
at the end of 2-min rest period (Fig.
S12D). Application of tetracaine increased SR Ca
2+
content
(3.96 ± 0.29, n
cells
= 21 vs 2.82 ± 0.28, n
cells
= 17, p≤ .01). Self-
ratioed [Ca
2+
]
i
at the end of 2-min rest period decreased with tetra-
caine (0.92 ± 0.04 vs 1.09 ± 0.06, p≤ .05).
Amplitudes of [Ca
2+
]
i
transients in response to the 0.5 Hz pacing de-
creased in NRVMs infected with TRPC1-eGFP (1.72 ± 0.09, n
cells
= 49)
compared to eGFP (2.19 ± 0.17, n
cells
= 34, P≤ .05) and shRNA-TRPC1-
eGFP (2.42 ± 0.24, n
cells
= 14, P ≤ .01). Differences between eGFP and
shRNA-TRPC1-eGFP infected cells were not significant.
3.5. Measurement of [Ca
2+
]
i
and SR Ca
2+
content in rabbit ventricular
myocytes
Using epifluorescence microscopy and a Ca
2+
sensitive dye, we
evaluated the decay of [Ca
2+
]
i
and the SR Ca
2+
content in rabbit
ventricular myocytes bathed in Na
+
and Ca
2+
free solution in absence
and presence of SKF-96365. We observed a slow decay of the Ca
2+
signal in quiescent cells in the absence of SKF-96365 (Fig. 8A), which is
commonly explained by SR uptake and sarcolemmal leak of cytosolic
Ca
2+
. Application of SKF-96365 intensified the Ca
2+
signal decay
versus control (−14.8% vs. -9.4%, n
cells
= 9, P < .05) (Fig. 8B and C).
The SR Ca
2+
content in the presence of SKF-96365 was slightly ele-
vated (1.50 ± 0.26 versus 1.76 ± 0.26, +17.3%, n
cells
= 6,
P < .05) (Fig. 8D). Peak systolic Ca
2+
signals during action potentials
just before switching to Na
+
and Ca
2+
free solution were similar in the
control and SKF-96365 group (Fig. S13), indicating that the SR Ca
2+
content was also similar at that time.
3.6. Modeling of SR Ca
2+
leak and effects on [Ca
2+
]
i
in NRVMs
A mathematical model of NRVM electrophysiology [33] was mod-
ified to qualitatively reproduce our measurements of SR Ca
2+
content.
Applying the protocol from our studies on NRVMs (Fig. 7A-C) and
varying SR Ca
2+
leak, the model revealed a negative relationship be-
tween SR Ca
2+
leak and release. The model reproduced experimental
differences (Fig. 7D) of caffeine-induced ∆[Ca
2+
]
i
due to TRPC1 si-
lencing with a 98% decrease in leak and due to TRPC1 overexpression
with 331% leak (Fig. S14B-E). The model also qualitatively predicted
the decrease of [Ca
2+
]
i.
Amplitudes in TRPC1-eGFP infected versus
eGFP and shRNA-TRPC1-eGFP NRVMs that we found in our experi-
mental studies.
3.7. Modeling of SR Ca
2+
leak and physiological effects on [Ca
2+
]
i
in
rabbit ventricular myocytes
We used computational modeling to shed light on the role of TRPC1
channels in electrophysiology and Ca
2+
signaling of rabbit myocytes at
physiological pacing rates. Measured cytosolic Ca
2+
decay in quiescent
rabbit ventricular myocytes in the presence of SKF-96365 (Fig. 8C) was
reconstructed by setting K
SR,TRPC
to −1.1 × 10
−5
/ms (Fig. S15A and
B). The faster [Ca
2+
]
i
decay in the presence of SKF-96365 was ac-
companied by a slowed decay of [Ca
2+
]
SR
(Fig. S15C and D). After two
minutes decay, [Ca
2+
]
SR
was elevated with SKF-96365 vs. control (at
end of pacing: 0.52 mM, after 2 min: 0.50 mM vs. 0.48 mM, −4.0% vs%
vs. -8.8%). The model predicts increased [Ca
2+
]
SR
after SKF-96365
application (+5.2%), which is consistent with increased measured SR
Ca
2+
content (Fig. 8D).
We compared [Ca
2+
]
i
transients in control cells, cells in presence of
SKF-96365 and cells with increased expression of TRPC1 at pacing rates of
2, 3 and 4 Hz (Fig. 9A). We assumed that increased expression (or activa-
tion) of TRPC1 channels leads to an increase of SR Ca
2+
leak to 400%
versus control levels. Thus, effects of upregulated TRPC1 channels were
reconstructed by increasing SR Ca
2+
leak (K
SR,TRPC
= 4.4 × 10
−5
/ms).
For all cell models, minimal (diastolic) and maximal (systolic)
[Ca
2+
]
i
increased with increasing pacing rate (Fig. 9B and C). For all
pacing rates, SR Ca
2+
leak exhibited a positive relationship with ex-
trema and the amplitude of the [Ca
2+
]
i
transient. Modulation of the
diastolic [Ca
2+
]
i
was strongest (+23.1%) for high SR Ca
2+
leak and
low pacing rate. Modulation of the systolic [Ca
2+
]
i
and amplitude was
strongest (+7.4% and 3.3%, respectively) for high SR Ca
2+
leak and
high pacing rate.
Similarly, [Ca
2+
]
SR
increased with pacing rates for all cell models
(Fig. 9D). While SR Ca
2+
leak exhibited a positive relationship with
[Ca
2+
]
SR
minima (Fig. 9E), the relationship with [Ca
2+
]
SR
maxima was
negative (Fig. 9F).
The effect of SR Ca
2+
leak on action potentials was small (Fig. 9G and
H). The action potential duration exhibited a negative relationship with
pacing rate. Increased SR Ca
2+
leak was associated with marginally in-
creased action potential duration at 90% repolarization (APD
90
) (Fig. 9I).
Fig. 7. Measurement and analysis of Ca
2+
transients in NRVMs using confocal microscopy. Self-ratioed Ca
2+
signals (F/F
0
) from a cell infected with (A) eGFP, (B)
TRPC1-eGFP and (C) shRNA-TRPC1-eGFP constructs. SR Ca
2+
content was assessed using rapid application of caffeine (20 mM). (D) Statistical analysis of SR Ca
2+
release. Cells overexpressing TRPC1 and after silencing exhibited a decreased and increased amplitude of the self-ratioed Ca
2+
signal ΔF
Caff
/F
0
, respectively, versus
control. Brackets mark significant differences (P < .05).
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
119
Fig. 8. Epifluorescence microscopy of rabbit myocyte using a cytosolic Ca
2+
indicator. Self-ratioed Ca
2+
signals (F/F
0
) are presented for a cell undergoing pacing,
followed by bathing with Na
+
and Ca
2+
free solution (A) without and (B) with the TRPC channel blocker SKF-96365. In this cell, F/F
0
decreased in Na
+
and Ca
2+
free solution. Subsequently, caffeine (20 mM) was rapidly applied. (C) Decay of F/F
0
after 2 min in Na
+
and Ca
2+
free solution was increased in the presence of SKF-
96365 (SKF) versus control (CTR). (D) Application of caffeine in cells bathed in SKF-96365 caused an increased amplitude of the self-ratioed Ca
2+
signal ΔF
Caff
/F
0
versus control reflecting an increased SR Ca
2+
content. Brackets mark significant differences (P < .05).
Fig. 9. Effects of SR Ca
2+
leak current investigated in a computational model of rabbit ventricular myocytes. Simulations were performed at a pacing rate of 2, 3 and
4 Hz. Block (SKF-96365) and increased expression (400%) of TRPC1 channels were modeled by decreased and increased SR Ca
2+
leak, respectively. (A) SR Ca
2+
leak
affected [Ca
2+
]
i
at physiological pacing rates. In particular, SR Ca
2+
leak exhibited a positive relationship with (B) minimal and (C) maximal [Ca
2+
]
i
. (D) [Ca
2+
]
SR
transients with associated (E) minima and (F) maxima. While SR Ca
2+
leak exhibited a positive relationship with the minimal [Ca
2+
]
SR
, the relationship with
maximal [Ca
2+
]
SR
was negative. (G,H) SR Ca
2+
leak had only a marginal effect on V
m
. (I) In particular, variation of SR Ca
2+
leak led to small differences of APD
90
versus control.
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
120
4. Discussion
Our studies provide insights into the subcellular location and
functional role of TRPC1 in ventricular cardiomyocytes. Previous stu-
dies on cardiomyocytes suggested that TRPC1 are localized in the sar-
colemma and involved in membrane electrophysiology and mechano-
electrical coupling. Our studies using immunolabeling and confocal
microscopy revealed an intracellular distribution of TRPC1 in ven-
tricular myocytes isolated from neonatal rat and adult rabbit hearts.
TRPC1 was colocalized with SERCA2, an established marker of SR
(Figs. 1, 2 and 5). Quantitative colocalization analysis based on Pear-
son's correlation coefficient calculated from 3D microscopic image
stacks further supported intracellular expression of TRPC1 adjacent to
Z-lines in adult rabbit myocytes. TRPC1 exhibited high colocalization
with sarcomeric α-actinin, which is a marker of the Z-line (Figs. 5E and
I). The degree of colocalization with WGA, an established marker of
sarcolemma, was significantly lower (Figs. 4 and 5I) and comparable to
colocalization of WGA with α-actinin, desmin and tubulin (Fig. 5J),
which are cytoskeletal proteins not located in the sarcolemma. Due to
limited spatial resolution of confocal microscopy, our approach cannot
completely exclude sarcolemmal expression of TRPC1. To address this
issue, we employed immunoelectron and super-resolution microscopy
with a spatial resolution in the nanometer range. For NRVMs, super-
resolution microscopy reproduced the striated intracellular pattern of
TRPC1 in proximity to SERCA2 (Fig. 3) as observed with confocal mi-
croscopy (Fig. 1). Electron microscopy of rabbit ventricular myocytes
supported the absence of TRPC1 expression in the outer sarcolemma
and t-system (Fig. S8). The images indicate a discrete intracellular
distribution with partial clustering of TRPC1 adjacent to Z-lines. While
our approach for immunoelectron microscopy did not identify the in-
tracellular compartment where the TRPC1 reside, the majority of
TRPC1 was found in or close to z-lines as well as within sarcomeres.
The SR membrane has a high density in the region of Z-lines in adult
rat and sheep myocytes [34]. Accordingly, in our studies using confocal
microscopy on adult rabbit ventricular myocytes we found an increased
SERCA2 fluorescence associated with the Z-line. The colocalization of
TRPC1 with SERCA2 and the striated pattern of TRPC1 support our
hypothesis that TRPC1 is located in the SR membrane. We note that the
cultured NRVMs used in our study do not exhibit a t-system, yet the
striated organization of native TRPC1 (Fig. 1A) was similar to the
TRPC1 organization in adult rabbit myocytes. Furthermore, regions of
rabbit myocytes devoid of t-tubules still showed a prominent TRPC1
signal (Fig. 4). This suggests that TRPC1 localization is independent of
sarcolemmal organization.
To provide further evidence for a SR location of TRPC1 channels in
ventricular myocytes, we investigated the relationship between TRPC1
expression and SR Ca
2+
content in quiescent NRVMs held in Na
+
and
Ca
2+
free solution for 2 min. SR Ca
2+
leak was assessed by measuring
SR Ca
2+
content. We found that TRPC1 expression exhibited a negative
relationship with SR Ca
2+
content: expression of TRPC1-eGFP construct
decreased the SR Ca
2+
content in NRVMs, while silencing of TRPC1
with shRNA-TRPC1-eGFP increased SR Ca
2+
content (Fig. 7). These
results were qualitatively reproduced in a mathematical model with
increased and decreased SR Ca
2+
leak corresponding to TRPC1 upre-
gulation and silencing, respectively (Fig. S14). Furthermore, applica-
tion of 5 μM SKF-96365, an established blocker of TRPC channels, in-
creased the SR Ca
2+
content in TRPC1-eGFP infected NRVMs (Fig.
S10). Since the experiments were performed in Ca
2+
free bathing so-
lution and Ca
2+
cannot enter the cells from the extracellular space, the
source of increased [Ca
2+
]
i
after caffeine application must be in in-
tracellular pools. The action of SKF-96365 to increase SR Ca
2+
content
points at the SR as the intracellular Ca
2+
pool, which supports our
imaging results. Blocking RyR leak with tetracaine (Fig. S12) led to
similar increase in SR Ca
2+
content as measured after silencing of
TRPC1, using our shRNA-TRPC1-eGFP construct (Fig. S12C vs. 7D).
Increased SR Ca
2+
content was associated with a decrease in [Ca
2+
]
i
at
the end of the 2 min rest period (Fig. S12D). The similarity of effects of
TRPC1 silencing and RyR block indicates that both localize to the same
organelle. Collectively, these results suggest that TRPC1 channels
contribute to Ca
2+
leak from the SR into the cytosol.
Cultured NRVMS are in many aspects different than native ven-
tricular myocytes, so we furthered our studies using adult rabbit ven-
tricular myocytes. RNA sequencing suggested that TRPC1 is the most
expressed member of the TRPC family in these cells (Fig. S7).
Application of SKF-96365 increased the decay of [Ca
2+
]
i
and the SR
Ca
2+
content in cells bathed in Na
+
and Ca
2+
free solution (Fig. 8). As
for NRVMs (Fig. S10), we explain this finding by block of TRPC1
channels in the SR and a subsequent reduction in SR Ca
2+
leak.
Our studies provide evidence for the contribution of TRPC1 chan-
nels to Ca
2+
leak from the SR into the cytosol in cardiomyocytes. SR
Ca
2+
leak in these cells has been explained by spontaneous sparks from
RyRs, non-spark-mediated RyR leak and non-RyR leak [24,26]. The
contribution of spark-mediated to total leak is thought to be small at
low SR Ca
2+
concentration, but large for high SR Ca
2+
concentrations.
Non-RyR leak was suggested to amount to around 50% of the non-
spark-mediated RyR leak. Knowledge on the structural basis of non-RyR
leak is sparse. A 1,4,5-inositol-trisphosphate (InsP
3
) dependent SR Ca
2+
leak through InsP
3
receptors has been identified, but was found to be
very low in the absence of InsP
3
. Also, non-RyR leak was insensitive to
block of the InsP
3
receptor. Based on our studies, we suggest that the
enigmatic non-RyR leak is caused, at least in part, by fluxes through SR
TRPC1 channels.
Our computational simulations using a mathematical model of
rabbit ventricular myocytes with the addition of TRPC1 channels in the
SR suggest that TRPC1 channels modulate Ca
2+
transients at physio-
logical pacing rates (Fig. 9). We assumed that TRPC1 channels allow
Ca
2+
to leak from the SR into the cytosol, which was described in a
similar manner as Ca
2+
leak through RyR channels already in-
corporated in this model. In our model, the leak was proportional to the
difference between [Ca
2+
]
SR
and [Ca
2+
]
i
, and thus dominated by
[Ca
2+
]
SR
, which is approximately three orders of magnitude larger than
[Ca
2+
]
i
. In the simulations we focused on sub-acute effects of TRPC1
channel activation at a similar time scale as in our experimental as-
sessment of [Ca
2+
]
i
. Increased SR Ca
2+
leak augmented [Ca
2+
]
i
during
all phases of action potentials at physiological pacing rates. In parti-
cular, diastolic and systolic [Ca
2+
]
i
were increased by TRPC1 leak.
Interestingly, effects of TRPC1 channels on action potentials were small
with only marginal prolongation of APD
90
. The prolongation is ex-
plained by increased [Ca
2+
]
i
causing increased forward so-
dium‑calcium exchanger activity, which is electrogenic. The prolonga-
tion also causes increased Ca
2+
influx through L-type Ca
2+
channels,
which contributes to the increase of [Ca
2+
]
i
. Based on these simula-
tions, we propose a physiological role for TRPC1 channels in the SR of
cardiac myocytes in modulation of [Ca
2+
]
i
and thus contractility. In
particular, the simulations suggest that leak Ca
2+
through TRPC1
channels increases contractility in paced myocytes.
Our localization study on TRPC1 in NRVMs and rabbit ventricular
myocytes is in conflict with previous studies on rodent cardiomyocytes,
many of which used confocal microscopy [8]. It is, however, difficult to
describe spatial relationships of the t-system and proteins in rodent
using confocal microscopy. Compared to rabbit, the t-system in rodents
is denser and tubules are of smaller diameter, which hinders in-
vestigations using confocal microscopy due to limitations of spatial
resolution [35,36]. To illustrate the issue, we imaged ventricular
myocytes from adult rat using a similar experimental approach as for
ventricular myocytes of rabbits (Supplementary Methods, Fig. S16).
TRPC1 did not outline the outer sarcolemma. However, the dense t-
system obscured localization of TRPC1.
Similar discrepancies regarding TRPC1 location emerged in studies
of skeletal myocytes. In a study on mouse skeletal muscle, Gervasio
et al. reported sarcolemmal expression of TRPC1 [37], whereas Stiber
et al. described a sarcolemmal pattern of TRPC1 corresponding to
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
121
costameres at the level of Z discs [38]. In contrast, Berbey et al. pre-
sented a striated pattern of TRPC1 that matched SERCA1 im-
munolabeling in mouse skeletal myocytes [23]. The study applied im-
munolabeling for endogenous TRPC1 and overexpressing TRPC1
conjugated with yellow fluorescent protein, which revealed an in-
tracellular distribution of TRPC1 similar to our findings in cardio-
myocytes.
Previous studies on oocytes and CHO cells suggested that TRPC1
constitute stretch-activated ion channels [10], which is however still
controversially discussed [39,40]. Based on those studies, a role in
membrane electrophysiology and mechano-electrical coupling of myo-
cytes was proposed. While our studies did not provide evidence for a
direct role of TRPC1 in membrane electrophysiology, the studies shed
new light on mechanisms by which TRPC1 may contribute to mechano-
electrical coupling. The mechano-sensitivity of TRPC1 channels may
contribute to stretch-modulated SR Ca
2+
leak.
Another topic that we attempted to address in this study is the
spatial relationship between TRPC1 and cytoskeleton in cardiomyo-
cytes. The cytoskeleton plays a critical role in anchoring, trafficking and
functionally regulating TRPC channels [31]. Also, Ca
2+
influx through
TRPC channels was suggested to be involved in rearrangement and
remodeling of the cytoskeleton in many cell types [32]. Our confocal
microscopic images revealed that TRPC1 is highly colocalized with
sarcomeric α-actinin, and to some degree with desmin and vinculin
along the Z-lines, whereas the distribution of TRPC1 appeared to be
perpendicular to microtubules. Although the degree of functional co-
localization with cytoskeletal proteins is unknown, we suggest that the
intimate relationship with the sarcomeres allows TRPC1 channels to
sense mechanical strain in the cell interior.
Our studies provide insights into the functional role of TRPC1, thus
it is tempting to extrapolate our findings towards understanding the
pathophysiological role of TRPC1 channels. It was suggested that
TRPC1 channels are critical players in cardiac hypertrophy and heart
failure [41]. Several studies indicated pathological up-regulation of
TRPC1 [4,42]. For example, hypertrophic agents such as endothelin-1
were found to increase TRPC1 expression to 410% in NRVMs. A simple
prediction from our findings and reported pathological up-regulation of
TRPCs is that increased Ca
2+
leak from the SR into the cytosol causes
sustainedly increased [Ca
2+
]
i
. Sustained increase of [Ca
2+
]
i
has been
implicated previously in hypertrophic signaling in cardiac myocytes
[43]. Recent studies indicate that TRPC channels are potential targets
for treating heart failure [7] and cardiac dystrophy [44]. Based on our
findings the mechanism of TRPC blockade as a way to treat cardio-
vascular disease would be explained by reduction of SR Ca
2+
leak
leading to normalization of [Ca
2+
]
i
, and inhibition of hypertrophic
signaling.
Our interpretation of the presented studies assumes that TRPC1
forms channels that conduct Ca
2+
. Extensive prior work revealed that
TRPC1 forms heteromeric channels with other members of the TRPC,
TRPP and TRPV families [39]. This suggests that modulation of TRPC1
expression as performed in our studies will, beyond modulation of the
density of homomeric TRPC1 channels, modulate the density of het-
eromeric channels and contribution of TRPC1 to those channels. Re-
cently, it was hypothesized that TRPC1 acts as regulatory subunit in
heteromeric channel complexes [39]. Further studies will be required to
understand effects of modulation of TRPC1 expression and interpret our
findings in the context of this hypothesis.
Limitations of our approach include the spatial resolution of con-
focal microscopy [36]. Limited resolution exacerbates assessment of
colocalization of proteins. Another limitation is related to our approach
for immunoelectron microscopy, which did not provide direct in-
formation on SR membrane.
We note the high variability of results using NRVMs, which is, in
part, explained by the variable degree of expression of adenoviral
constructs. Even beyond variable expression of adenoviral constructs,
we noticed large variability in our studies on NRVMs. For instance,
eGFP expressing NRVMs exhibited a large variability of SR Ca
2+
con-
tent. This explains why application of SKF-96365 in these cells did not
yield statistically significant differences (Fig. S10). The high variability
complicates testing of statistical hypotheses. For instance, a-priori
power analysis based on preliminary studies on cells infected with eGFP
with and without SKF-96365 application suggested that a large sample
size (n~1000) is required for statements on statistically significant
differences between the two groups.
High variability in NRVM phenotypes also limits accuracy of the
mathematical model. The original model was constructed from ex-
periments in various laboratories where culture conditions were pre-
sumably heterogeneous and different to the conditions in our labora-
tory. Notably, temperature differences between the model and our
experiments restrict direct comparison. The original model was devel-
oped from measurements with NRVMs at 32 °C, while our experiments
were performed at room temperature (~22 °C). We applied room
temperature in order to control pacing rate of the cells, but temperature
affects many cellular functions. Thus, the simulations served primarily
for a qualitative comparison with our experimental findings. A general
limitation of our models is related to modeling of caffeine effects. We
limited our modeling of NRVMs to the initial phase of Ca
2+
release (Fig.
S14D) relevant for comparison with our measurements. Further work,
e.g. accounting for wash-in and out of caffeine, will be necessary to
accurately model the decay phase.
We also note differences of the spatial distribution on native TRPC1
and TRPC1-eGFP (Fig. 2H). We speculate that different time scales for
expression and localization of the native TRPC1 and TRPC1-eGFP ex-
plain the differences. A significant amount of the TRPC1-eGFP construct
may be still trafficking to the sites, where the native TRPC1 resides.
Nevertheless, similar R
r
values for SERCA2 with native TRPC1 (Fig. 2J)
and TRPC1-eGFP (Fig. 2K) construct suggest trafficking of the construct
to SR regions. The TRPC1-eGFP vector comprises fused eGFP molecule
yielding fluorescence upon expression of the TRPC1 protein (Fig. 6B).
In contrast, the eGFP and shRNA-TRPC1-eGFP vectors comprise a non-
fused eGFP yielding fluorescence upon successful infection of the cell.
Images from living NRVMs infected with eGFP and shRNA-TRPC1-eGFP
constructs indicate cytosolic and nuclear localization of the constructs
(Fig. 6A and C), likely due to intracellular diffusion of the small non-
fused eGFP molecule.
We acknowledge limitations of our functional studies related to the
absence of specific blockers or activators for TRPC1 channels. These
limitations triggered our development and application of adenoviral
TRPC1 overexpression and silencing in the presented studies on
NRVMs. Nevertheless, in some studies we applied SKF-96365, which is
an unspecific TRPC channel blocker, which also affects TRPV2, TRPM8
and voltage-gated calcium (Ca
v
) 1–3 channels [45]. We accounted, in
part, for the weak specificity of SKF-96365 with our experimental
protocol. SKF-96365 was applied to myocytes after pacing and during
rest for 2 min. The membrane voltage of myocytes at rest is close to the
Nernst voltage for potassium, and thus voltage gated channels, such as
Ca
V
1–3, are closed and not involved in our measurements. Further in-
sights into potential effects of unspecific action of SKF-96365 arise from
our RNASeq data from rabbit ventricular myocardium. These data
suggest that expression of TRPC1 is much higher than expression of
TRPC 3, 4, 6 and 7 (Fig. S7) as well as TRPV2 and TRPM8 [29]. While
we cannot fully exclude that SKF-96365 effects on these channels affect
our measurements, we note that the results from application of SKF-
96365 (Figs. 8D and S12) are consistent with results applying TRPC1
silencing (Fig. 7).
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.yjmcc.2020.01.008.
Author contributions
Designed research: QH, AAA, KWS, FBS.
Performed research: QH, AAA, MS, KWS, CH, LN.
Q. Hu, et al. Journal of Molecular and Cellular Cardiology 139 (2020) 113–123
122
Analyzed data: QH, AAA, TS, MS, FBS.
Wrote the manuscript: QH, AAA, MS, FBS.
Sources of funding
We acknowledge funding by the Nora Eccles Treadwell Foundation
and the National Heart, Lung and Blood Institute (R01HL094464 and
R01HL132067).
Disclosures
None declared.
Acknowledgements
We acknowledge molecular biology support and expert advice on
adenoviral expression of the TRPC1 constructs from Dr. Michael
Sanguinetti. We thank Dr. Boris Martinac for providing us with the
TRPC1 construct. We also thank Mr. Brett Milash for his help with the
RNASeq data analysis. We appreciate the support of Dr. Kate Larson,
who provided us with tissue samples from WT and TRPC1 KO mice.
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