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Canonical Transient Receptor Potential Channels Promote
Cardiomyocyte Hypertrophy through Activation
of Calcineurin Signaling
*
Received for publication, June 9, 2006, and in revised form, August 31, 2006 Published, JBC Papers in Press, September 1, 2006, DOI 10.1074/jbc.M605536200
Erik W. Bush
‡1
, David B. Hood
‡
, Philip J. Papst
‡
, Joseph A. Chapo
‡
, Wayne Minobe
§
, Michael R. Bristow
§
,
Eric N. Olson
¶2
, and Timothy A. McKinsey
‡3
From
‡
Myogen, Inc., Westminster, Colorado 80021, the
§
Division of Cardiology, University of Colorado Health Sciences Center,
Denver, Colorado 80262, and the
¶
University of Texas Southwestern Medical Center, Dallas, Texas 75390
The calcium/calmodulin-dependent phosphatase calcineurin
plays a central role in the control of cardiomyocyte hypertrophy
in response to pathological stimuli. Although calcineurin is
present at high levels in normal heart, its activity appears to be
unaffected by calcium during the course of a cardiac cycle. The
mechanism(s) whereby calcineurin is selectively activated by
calcium under pathological conditions has remained unclear.
Here, we demonstrate that diverse signals for cardiac hypertro-
phy stimulate expression of canonical transient receptor poten-
tial (TRPC) channels. TRPC consists of a family of seven mem-
brane-spanning nonselective cation channels that have been
implicated in the nonvoltage-gated influx of calcium in response
to G protein-coupled receptor signaling, receptor tyrosine
kinase signaling, and depletion of internal calcium stores.
TRPC3 expression is up-regulated in multiple rodent models of
pathological cardiac hypertrophy, whereas TRPC5 expression is
induced in failing human heart. We demonstrate that TRPC
promotes cardiomyocyte hypertrophy through activation of cal-
cineurin and its downstream effector, the nuclear factor of acti-
vated T cells transcription factor. These results define a novel
role for TRPC channels in the control of cardiac growth, and
suggest that a TRPC-derived pool of calcium contributes to
selective activation of calcineurin in diseased heart.
Cardiac hypertrophy is an adaptive response of the heart to
many forms of cardiac disease, including hypertension,
mechanical load abnormalities, myocardial infarction, valvular
dysfunction, cardiac arrhythmias, endocrine disorders, and
genetic mutations in cardiac contractile protein genes. Whereas
the hypertrophic response is thought to be an initially compensa-
tory mechanism that augments cardiac performance, sustained
hypertrophy is maladaptive and frequently leads to ventricular
dilation and the clinical syndrome of heart failure. Accordingly,
cardiac hypertrophy has been established as an independent risk
factor for cardiac morbidity and mortality (1).
Abnormal calcium handling, characterized by elevated intra-
cellular diastolic calcium levels, is a hallmark of cardiac hyper-
trophy and heart failure. Elevated intracellular calcium not only
impairs the contractile performance of the heart, but also acti-
vates calcium-dependent signaling pathways that mediate mal-
adaptive cardiac remodeling (2). One such pathway is regu-
lated by the calcium-calmodulin-dependent phosphatase
calcineurin, which has been shown to be sufficient, and in some
cases, necessary for pathological hypertrophy (3–5). Activated
calcineurin dephosphorylates the transcription factor nuclear
factor of activated T-cells (NFAT),
4
facilitating translocation of
NFAT to the nucleus where it acts in concert with other pro-
teins to mediate expression of prohypertrophic genes. The
activity of calcineurin is tightly regulated in vivo by a negative
feedback mechanism; one of the most highly sensitive NFAT
target genes encodes a potent calcineurin inhibitor, modulatory
calcineurin-interacting protein 1 (MCIP1) (6–8), also known
as Down syndrome critical region 1.
How pathological stress is translated into a calcium stimulus
capable of activating cardiac calcineurin, and why calcineurin
is spared from activation in response to the calcium that drives
muscle contraction, remain important unanswered questions.
In lymphocytes, influx of extracellular calcium through calcium
release-activated calcium channels provides the sustained cal-
cium signal required to activate the calcineurin/NFAT pathway
(9). Unlike voltage-gated L-type calcium channels, calcium
entry via calcium release-activated calcium channels is trig-
gered by the release of calcium from intracellular stores, a
mechanism referred to as store-operated calcium or capacita-
tive calcium entry. Recent work has confirmed that hyper-
trophic agonists stimulate capacitative calcium entry in cardiac
myocytes, leading to activation of the calcineurin/NFAT path-
way and cardiac hypertrophy (10, 11). However, the identity of
* The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence may be addressed. Tel.: 303-464-5234; Fax: 303-
410-6669; E-mail: erik.bush@myogen.com.
2
Supported by grants from the National Institutes of Health, The Donald W.
Reynolds Clinical Cardiovascular Research Center, The Robert A. Welch
Foundation, and the Texas Advanced Technology Program.
3
To whom correspondence may be addressed. Tel.: 303-533-1736; Fax: 303-
410-6669; E-mail: timothy.mckinsey@myogen.com.
4
The abbreviations used are: NFAT, nuclear factor of activated T-cells; MCIP1,
modulatory calcineurin-interacting protein 1; TRPC, canonical transient
receptor potential channels; PAMH, pyridine activator of myocyte hyper-
trophy; BTP2, N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-
methyl-1,2,3-thiadiazole-5-carboxamide; LV, left ventricle; OAG, oleoyl-2-
acetyl-sn-glycerol; 2APB, 2-aminoethoxydiphenylborane; GFP, green
fluorescent protein; NRVM, neonatal rat ventricular myocyte; DMEM, Dul-
becco’s modified Eagle’s medium; SHHF, spontaneous hypertensive heart
failure; TAB, thoracic aortic banding; PBS, phosphate-buffered saline; RT,
reverse transcriptase; ANF, atrial natriuretic factor.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 44, pp. 33487–33496, November 3, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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the cardiac channel(s) responsible for capacitative calcium
entry remains unknown.
Members of the canonical transient receptor potential
(TRPC) family of channels have been implicated in the control
of capacitative calcium entry in non-cardiac cell types (12). The
seven members of the TRPC family (TRPC1–7) are nonselec-
tive calcium-permeable cation channels. Most family members
can be activated directly by diacylglycerol, a second messenger
product of phospholipase C. Furthermore, TRPC channels
have recently been shown to function as stretch receptors,
admitting calcium influx in response to mechanical stress (13,
14), a pathological stimulus known to contribute to the devel-
opment of cardiac hypertrophy (15). Other signaling molecules
known to regulate TRPC channel activity include the receptor
tyrosine kinase Src (16) and the cGMP-dependent protein
kinase PKG (17).
Previously, we reported results of microarray studies with
RNA from cultured cardiac myocytes exposed to distinct
hypertrophic stimuli (18). Among the genes most potently up-
regulated by two distinct hypertrophic agonists, the
␣
-adrener-
gic receptor agonist, phenylephrine (PE), and the serotonin
receptor agonist, pyridine activator of myocyte hypertrophy
(PAMH), was that encoding TRPC3. Here we demonstrate that
cardiacTRPC3expressionisup-regulatedthroughacalcineurin-
dependent mechanism by discrete pharmacologic, genetic, and
physiologic stimuli that trigger pathological cardiac hypertro-
phy in rodent models. In contrast, TRPC5 is selectively induced
in failing human heart. Gain and loss of function studies dem-
onstrate that TRPC channels positively regulate cardiac hyper-
trophy through activation of calcineurin/NFAT signaling.
These results suggest a role for TRPC channels in the transmis-
sion of pathological calcium signals in the heart.
EXPERIMENTAL PROCEDURES
Chemical Reagents, Plasmids, and Adenoviral Constructs—PE,
endothelin-1, oleoyl-2-acetyl-sn-glycerol (OAG), and 2-aminoe-
thoxydiphenylborane (2-APB) were obtained from Sigma. N-{4-
[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-
thiadiazole-5-carboxamide (BTP2) was obtained from Calbiochem.
The cDNA encoding full-length, human TRPC3 (accession number
NM_003305) was cloned by reverse transcriptase-PCR with the One
Step RT-PCR kit (Invitrogen). For adenovirus production, this cDNA
was subcloned into pShuttle-cytomegalovirus (QBiogen) and adeno-
virus was generated in HEK293 cells according to the manufacturer’s
recommendations. Clonal populations of virus were obtained using
the agar overlay method and titered with the Adeno-X Rapid
Titer Kit (Clontech). The adenovirus encoding GFP-NFATc
(NFAT2) was provided by Dr. Martin Schneider (University of
Maryland, Baltimore, MD).
Cardiomyocyte Culture—Neonatal rat ventricular myocytes
(NRVMs) were prepared from 1–2-day-old Sprague-Dawley
rats as described previously (19). Cells were plated on gelatin-
coated plates in DMEM containing fetal bovine serum (10%),
penicillin-streptomycin, and
L-glutamine (DMEM complete).
After 12 h, plating medium was replaced with serum-free
DMEM supplemented with Nutridoma-SP (0.1%; Roche
Applied Sciences), which contains albumin, insulin, transferrin,
and other defined organic and inorganic compounds the fol-
lowing day. Cells were treated with agonists/inhibitors for
48–72 h.
Animal Models and Human Heart Samples—The Institu-
tional Animal Care and Use Committee approved all animal
protocols. The spontaneous hypertensive heart failure (SHHF)
rat is a well documented genetic model of hypertension, cardiac
hypertrophy, and heart failure (20). For the isoproterenol
model, male Sprague-Dawley rats were implanted subcutane-
ously with Alzet Mini-Pumps filled with either vehicle control
(0.5 mg/ml sodium bisulfite in 0.9% sterile NaCl) or isoproter-
enol (dissolved in vehicle sufficient to administer 5 mg/kg/day
of isoproterenol for 4 days). For thoracic aortic banding (TAB),
male Sprague-Dawley rats (8 –9 weeks of age, 200 –225 g) were
anesthetized with 5% isoflurane (v/v 100% O
2
), intubated, and
maintained at 2.0% isoflurane with positive pressure ventila-
tion. A left thoracotomy through the third intercostal space was
performed and the descending thoracic aorta 3–4 mm cranial
to the intersection of the aorta and azygous vein was isolated. A
segment of 5-0 silk suture was then positioned around the iso-
lated aorta to function as a ligature. A blunted hypodermic nee-
dle (gauge determined by weight) was placed between the aorta
and the suture to prevent complete aortic occlusion when the
suture was tied. When tying was completed, the needle was
removed from between the aorta and ligature, re-establishing
flow through the vessel. The thorax was then closed and the
pneumothorax evacuated. After 7 days of recovery, animals
were sacrificed and left ventricular tissue processed for West-
ern blot analysis. Average heart weight to body weight ratios in
banded versus sham-operated rats increased 22% at 1 week. The
construction and characterization of transgenic mice express-
ing constitutively active calcineurin have been described else-
where (3).
Ventricular samples from human patients with end-stage idi-
opathic dilated cardiomyopathy or nonfailing human cardiac
tissue samples were obtained through the University of Colo-
rado Health Sciences Center Cardiac Transplantation Pro-
gram. End-stage failing hearts came from individuals who
underwent heart transplantation because of idiopathic dilated
cardiomyopathy (n ⫽ 6; 3 male, 3 female, average age 49.8
years). Nonfailing controls were obtained from organ donors
whose hearts were unsuitable for donation due to blood type or
size incompatibilities (n ⫽ 6; 4 male, 2 female; average age 51.8
years). Tissue samples were taken immediately upon explanta-
tion and rapidly frozen in liquid nitrogen.
Immunoblotting—NRVMs and in vivo tissue samples were
homogenized in Tris buffer (50 m
M, pH 7.5) containing EDTA
(5 m
M), Triton X-100 (1%), protease inhibitor mixture (Com-
plete, Roche), phenylmethylsulfonyl fluoride (1 m
M), and phos-
phatase inhibitors (sodium pyrophosphate, 1 m
M; sodium flu-
oride, 2 m
M;

-glycerolphosphate, 10 mM; sodium molybdate, 1
m
M; and sodium orthovanadate, 1 mM). Homogenates were
briefly sonicated and cleared by centrifugation. Protein concen-
trations were determined by BCA assay (Pierce) and 15
gof
total protein was resolved by SDS-PAGE with gradient gels
(4–20% polyacrylamide; Invitrogen). Proteins were transferred
to nitrocellulose membranes (Bio-Rad) and probed with
TRPC3- or TRPC5-specific antibodies, according to the manu-
facturer’s instructions (Alomone Labs). Experiments were per-
TRPC Channels Promote Cardiomyocyte Hypertrophy
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formed with freshly diluted antibodies, because in our experi-
ence we have found that the lyophilized antibodies, once
resuspended, lose significant immunoreactivity in storage.
Negative controls for specificity were performed by preincubat-
ing the antibody with the corresponding antigen peptide (0.75
g of peptide/1
g of antibody) for 1 h. Antibodies against
MCIP1 and calnexin have been described previously (18). Pro-
teins were visualized using an enhanced chemiluminescence
system (Pierce). For immunoprecipitation experiments, 3
gof
antibody was incubated with homogenate in a total volume of
0.5 ml for 1 h. Protein G-Sepharose beads (10
l, GE Health-
care) were added, and after a 1-h incubation, beads were
washed three times in homogenization buffer and prepared for
SDS-PAGE.
Hypertrophy Measurements—The atrial natriuretic factor
(ANF) competition enzyme-linked immunosorbent assay,
measurement of

-myosin heavy chain expression, RNA dot
blot assay for hypertrophic marker gene expression, and adeny-
late kinase release assay have been described previously (19).
For cell volume measurements, NRVM cultured in 6-well
dishes were harvested by treatment with trypsin (Cellgro).
After recovery by centrifugation, cell pellets were washed in
PBS, resuspended in 10 ml of IsoFlow electrolyte solution
(Beckman-Coulter), and analyzed with a Z2 Coulter Particle
Counter and Size Analyzer (Beckman-Coulter).
RT-PCR Analysis—RNA was isolated from NRVMs using
TRI Reagent威 (Sigma) according to the manufacturer’s instruc-
tions. RNA was quantified using both standard spectrophotom-
etry of nucleic acids at 260 nm using a DU64 spectrophotome-
ter (Beckman-Coulter) and fluorescence with a TD700
fluorometer (Turner Designs). RT-PCR of rat TRPC3 (acces-
sion number NM_021771) used primers that spanned the first
intron (7950 bp): forward primer, 5⬘-ctggccaacatagagaaggagt-
3⬘; and reverse primer, 5⬘-caccgattccagatctccat-3⬘. Rat 18 S
ribosomal RNA amplicons were used as input controls: forward
primer, 5⬘-cgaggaattcccagtaagtgc-3⬘, and reverse primer,
5⬘-aagttcgaccgtcttctcagc-3⬘. Reverse transcription and amplifi-
cation of 0.5
g of total RNA were done using the Super-
Script
TM
One-step RT-PCR System with Platinum威 Taq DNA
Polymerase (Invitrogen), on a I-cycler
TM
thermocyler (Bio-
Rad). Reverse transcription reactions were run on 2% agarose
gels containing 0.1
g/ml ethidium bromide. Imaging and den-
sitometry were performed with an Alpha Innotech gel imaging
system and ChemImage 5.5 software. RT-PCR amplified a sin-
FIGURE 1. Hypertrophic agonists stimulate TRPC3 expression in cultured cardiac myocytes. A, NRVMs (2 ⫻ 10
6
) were cultured in serum-free medium and
exposed to endothelin-1 (ET-1;50n
M), PE (20
M), or fetal bovine serum (FBS; 10%) for 72 h, as indicated. RT-PCR analysis was performed with primers specific
for TRPC3 or 18 S ribosomal RNA. B, TRPC3 mRNA levels were quantified by densitometry of ethidium bromide-stained gels and normalized to 18 S RNA to
control for differences in RNA input/loading. Values represent averages ⫾ S.D. from three independent sets of cells per condition. C, NRVMs were treated with
PE (20
M) in the absence or presence of cyclosporine A (CsA; 500 nM) or FK506 (1 nM). TRPC3 mRNA expression was analyzed as described in B. Values represent
averages ⫾ S.D. from three independent sets of cells per condition. D, total protein from NRVMs, rat left ventricle and mouse left ventricle was prepared and
analyzed by immunoblotting with a TRPC3-specific antibody. As a specificity control, the TRPC3 antibody was preincubated with the corresponding antigen
peptide. The immunoblot was exposed to film for 5 (left panel) and 30 s (right panel). E, NRVMs were treated with PE (20
M) as described in A. Total protein was
prepared and analyzed by immunoblotting with a TRPC3-specific antibody. Blots were reprobed with anti-calnexin antibodies to control for protein loading.
F, TRPC3 protein levels from E were quantified by densitometry. Values represent averages ⫾ S.D. from three independent sets of cells per condition. *, p ⬍ 0.01.
TRPC Channels Promote Cardiomyocyte Hypertrophy
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gle 141-bp band of the expected size, which sequencing
confirmed to be TRPC3. Negative control reactions excluding
RNA template, reverse transcriptase, or either primer did not
yield a PCR product. For analysis of human samples, RNA
was extracted from left ventricular tissue as described above
and subjected to TaqMan real-time quantitative RT-PCR
(PerkinElmer Life Sciences) as described by Riccio et al. (21).
Human TRPC5 primers were: forward, 5⬘-cctctcatcagaaccatgc-
caa-3⬘, and reverse, 5⬘-gcgtttgcttgatgactcagc-3⬘.
Indirect Immunofluorescence—NRVMs were plated on gela-
tin-coated 6-well dishes (2.5 ⫻ 10
5
cells/well) in DMEM com
-
plete and transferred 12 h subsequently to serum-free DMEM
supplemented with Nutridoma-SP (0.1%; Roche Applied Sci-
ences). Adenovirus was added to cells at the time of plating.
Unless otherwise indicated, virus was used at a multiplicity of
infection of 20. Following experimental treatment, cells were
washed two times in PBS, fixed with formalin (10%) in PBS,
permeabilized, and blocked with PBS containing Nonidet P-40
(0.1%) and bovine serum albumin (3%), and then incubated in
the same solution containing primary antibodies specific for
either sarcomeric
␣
-actinin (Sigma; 1:200 dilution) or Myc
(Santa Cruz; 1:500 dilution) for1hatroom temperature. Cells
were washed 4 times in PBS and incubated in PBS/Nonidet
P-40/bovine serum albumin containing either a fluorescein- or
CY3-conjugated secondary antibody (Jackson Laboratories;
1:200 dilution) for 30 min at room temperature. Cells were
washed 4 times in PBS and then covered with mounting solu-
tion (SlowFade, Molecular Probes) and glass coverslips. Pro-
teins were visualized with an inverted fluorescence micro-
scope (Olympus model BH-2) at ⫻40 magnification, and
images were captured using a digital camera (Photometrics;
Roper Scientific).
RESULTS
Calcineurin-dependent Induction of TRPC3 Expression in
Cultured Cardiac Myocytes—To gain insight into changes in
gene expression underlying stress-induced cardiac hypertro-
phy, RNA microarray studies were performed with cultured
NRVMs. NRVMs undergo hypertrophy in response to multiple
agonists, including PE, which stimulates the
␣
1
-adrenergic
receptor, and PAMH, a novel 5-HT2B serotonin receptor ago-
nist. A complete summary of microarray results with RNA from
NRVMs stimulated with PE or PAMH for 48 h is found in
the NCBI Gene Expression Omnibus (GEO) and are accessible
through GEO Series accession number GSE925. Among the
genes most potently up-regulated by both agonists were those
encoding

-myosin heavy chain, a classical marker of cardiac
hypertrophy, and the transient receptor potential channel 3
(TRPC3) calcium channel.
Although calcium is known to play a key role in the regula-
tion of cardiac hypertrophy (2), the relative contribution of dis-
tinct calcium pools to this growth response remains unclear. As
such, we focus here on elucidating the potential role of TRPC
channels in the control of stress-induced cardiac hypertrophy.
To confirm the microarray results, semi-quantitative
RT-PCR was performed with RNA from freshly isolated
NRVMs treated with PE or two independent hypertrophic ago-
nists, endothelin-1 and fetal bovine serum, for 48 h. Each ago-
nist stimulated expression of TRPC3 mRNA transcripts, with
PE and fetal bovine serum inducing the highest levels (Fig. 1, A
and B). Similar results were obtained with RNA from PAMH-
treated cells (data not shown).
Prior studies have suggested that signaling by the calcineurin
phosphatase stimulates expression of TRPC3 in skeletal muscle
FIGURE 2. Induction of cardiac TRPC3 protein expression in vivo. A, male,
2-month-old Sprague-Dawley (SD) rats were given a sham operation or sub-
jected to thoracic aortic banding to trigger pressure overload hypertrophy of
the LV of the heart, as described under “Experimental Procedures.” Seven
days following the operation, animals were sacrificed and TRPC3 levels in LV
protein lysates analyzed by immunoblotting. Blots were reprobed with cal-
nexin-specific antibodies to control for protein loading. B, parallel samples of
LV protein lysates were run to assess MCIP1 protein levels by immunoblot. C,
male SHHF rats of the indicated ages were sacrificed and TRPC3 levels in LV
preparations assessed as described in A. D, male SD rats were implanted with
osmotic mini-pumps that release either vehicle or the

-adrenergic receptor
agonist isoproterenol (ISO, 4 mg/kg/day) for 4 days. TRPC3 levels in LV prep-
arations were assessed as described in A. E, male mice (⬃2 months of age)
expressing a constitutively active calcineurin transgene (Cn-Tg) and control
nontransgenic littermates (WT) were sacrificed and TRPC3 and MCIP1 levels in
LV preparations assessed as described in A. F, TRPC3 protein levels in the
indicated models were quantified by densitometry. Values represent aver-
ages ⫾ S.D. *, p ⬍ 0.05.
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(22). To determine the role of cal-
cineurin in TRPC3 gene regulation
in cardiac myocytes, RT-PCR was
performed with RNA from NRVMs
stimulated with PE in the absence or
presence of the calcineurin inhibi-
tors cyclosporine A or FK506. As
shown in Fig. 1C, agonist-depend-
ent induction of TRPC3 mRNA
transcripts was completely blocked
by either of the two calcineurin
inhibitors.
To evaluate expression of TRPC3
protein expression in NRVMs and
rat and mouse ventricles, we per-
formed immunoblot analysis with a
TRPC3-specific antibody. As a con-
trol for specificity, the TRPC3 anti-
body was preincubated with the
antigen peptide. The antibody
detected immunoreactive bands of
the expected molecular mass
(⬃110–120 kDa) (Fig. 1D, left
panel). The most abundant band
observed in NRVM was ⬃115 kDa,
with a slightly smaller band of ⬃110
kDa predominating in rat and
mouse ventricular samples. Nota-
bly, detection of these immunoreac-
tive bands was efficiently competed
by preincubation of the TRPC3
antibody with the antigen peptide.
Intentional overexposure of the
same immunoblot (Fig. 1D, right
panel) revealed a number of non-
specific bands that were not com-
peted in the antigen peptide-blocked
controls. Immunoblot analysis veri-
fied that the observed induction of
TRPC3 mRNA correlated with mod-
est (between 1.5- and 2-fold) but sig-
nificant increases in TRPC3 protein
levels in PE-treated NRVMs (Fig. 1, E
and F).
Induction of TRPC3 Expression
in Rodent Models of Cardiac Hy-
pertrophy—Experiments were per-
formed to determine whether
TRPC3 expression is elevated in
animal models of pathological car-
diac hypertrophy. TAB places a
pressure overload on the heart and
triggers left ventricular (LV) hy-
pertrophy. Immunoblotting studies
revealed that TRPC3 protein ex-
pression was consistently elevated in
LVs of rats subjected to TAB for 7
days (Fig. 2A).
FIGURE 3. Ectopic TRPC3 stimulates cardiomyocyte hypertrophy. A, NRVMs (2 ⫻ 10
6
) were cultured in
10-cm dishes, and infected with Myc-tagged TRPC3 adenovirus (Ad-TRPC3) for 48 h. Total protein was prepared
and analyzed by immunoblotting with antibodies to TRPC3 (left panel, two independent clones shown) and the
Myc epitope tag (right panel). The apparent molecular mass of endogenous and recombinant TRPC3 was ⬃115
kDa. B, samples prepared as in panel A were immunoprecipitated with either the anti-Myc antibody or the
anti-TRPC3 antibody and immunoblotted with the other antibody, confirming that both antibodies immuno-
precipitated recombinant TRPC3. C, NRVMs were cultured in 6-well dishes (2.5 ⫻ 10
5
/well) and infected with
adenoviruses encoding

-galactosidase (Ad-

-GAL) or Myc-tagged TRPC3 (Ad-TRPC3). Cells were cultured for
48 h in serum-free medium in the absence or presence of the TRPC agonist OAG (100
M). Cells were fixed and
sarcomeres were visualized by indirect immunofluorescence with anti-sarcomeric actinin antibodies (top row).
Higher magnification images of the same cells are shown (middle row). Cells were co-stained with anti-Myc
antibody to reveal the subcellular distribution of TRPC3 (bottom row). D, NRVMs prepared as in panel B were
harvested by trypsinization, and mean cell volumes were determined by Coulter analysis. Values represent aver-
ages ⫾ S.D. for four independent sets of cells per condition. *, p ⬍ 0.01 versus uninfected control; #, p ⬍ 0.05
versus Ad-TRPC3 alone. E, NRVMs (2 ⫻ 10
6
) were cultured in 10-cm dishes, infected with either Ad-

-galactosidase or
Ad-TRPC3, and maintained in serum-free medium for 72 h. Total RNA was prepared and atrial natriuretic factor (ANF)
and
␣
-skeletal actin (
␣
-SK-actin) mRNA abundance determined by dot blot analysis with radiolabeled probes. Values
represent averages ⫾ S.D. for three independent sets of cells per condition. *, p ⬍ 0.05.
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To determine whether induction of TRPC3 expression cor-
relates with stimulation of calcineurin signaling, we quantified
expression of the endogenous NFAT target gene, modulatory
calcineurin-interacting protein-1 (MCIP1). The MCIP1 pro-
tein is a negative-feedback regulator of calcineurin signaling
(8), and is expressed as 28- and 38-kDa isoforms (18). Expres-
sion of 28-kDa MCIP1 is regulated by an alternative promoter
that harbors 15 NFAT binding sites, and thus this lower molec-
ular weight form of the protein serves as a sensitive marker of
NFAT activity (23). We observed significantly increased
expression of 28-kDa MCIP1 in TAB ventricles, indicating
enhanced calcineurin/NFAT activation (Fig. 2B). These obser-
vations are consistent with a role of calcineurin in the control of
TRPC3 gene expression.
SHHF rats provide a genetic
model of pathological cardiac
remodeling and heart failure (20).
Due to a mutation in the leptin
receptor gene, SHHF rats develop
hypertension that results in cardiac
hypertrophy and eventual heart fail-
ure. As shown in Fig. 2C, TRPC3
protein levels were elevated in LVs
from failing hearts of ⬃20-month-
old SHHF rats. However, TRPC3
expression was normal in 8–9-
month-old rats with compensated
LV hypertrophy. TRPC3 protein
expression was also increased in
LVs of rats infused for 4 days with
the
␣
-adrenergic receptor agonist
isoproterenol, which stimulates car-
diac hypertrophy that is accompa-
nied by fibrosis (Fig. 2D). Similar to
the TAB model, increased expres-
sion of MCIP1 protein was observed
in LV samples from both ISO-in-
fused and ⬃20-month-old SHHF
rats (data not shown).
To address the role of calcineurin
signaling in the control of cardiac
TRPC3 expression, TRPC3 protein
levels were measured in LVs of
transgenic mice expressing consti-
tutively active calcineurin in the
heart under the control of the
␣
-my-
osin heavy chain promoter (Cn-Tg)
(3). As shown in Fig. 2E, TRPC3
expression was dramatically ele-
vated in Cn-Tg mice compared with
wild-type littermates. Levels of
TRPC3 protein in LV samples from
each model of cardiac hypertrophy
were quantified by densitometry
(Fig. 2F). Together, the results dem-
onstrate that TRPC3 protein
expression is induced by diverse
cues for pathological cardiac growth
in vivo, and suggest a role for calcineurin signaling in the induc-
tion of cardiac TRPC3 expression.
Regulation of Cardiomyocyte Hypertrophy by TRPC Chan-
nels—Cardiac hypertrophy is associated with enhanced protein
synthesis, cell growth in the absence of mitosis, and increased
sarcomere assembly. In addition, fetal cardiac genes, including
those that encode ANF and
␣
-skeletal actin, and

-myosin
heavy chain become reactivated during the hypertrophic
response. To begin to address the role of TRPC channels in the
regulation of cardiac hypertrophy, we developed an adenovirus
that encodes full-length Myc epitope-tagged TRPC3 (Ad-
TRPC3) (Fig. 3A). As an additional control for antibody selec-
tivity, we determined that both anti-Myc and anti-TRPC3 anti-
bodies effectively immunoprecipitated recombinant TRPC3
FIGURE 4. Inhibition of hypertrophic gene expression by 2-APB and BTP2. NRVMs were cultured in 96-well
dishes and exposed to increasing doses of two different TRPC channel inhibitors, 2-APB or BTP2, in the absence
or presence of PE (20
M). After 48 h of stimulation, ANF abundance in culture supernatants, (A and B) cellular

-myosin heavy chain expression (C and D), and adenylate kinase release (E and F) (cytoxicity marker) were
measured as described under “Experimental Procedures.”
TRPC Channels Promote Cardiomyocyte Hypertrophy
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(Fig. 3B). NRVMs were infected with Ad-TRPC3 or control
virus expressing

-galactosidase (Ad-

-galactosidase) and cul-
tured for 48 h in the absence or presence of OAG, a diacylglyc-
erol analog that binds to and stimulates TRPC channel activity
(24, 25). As shown in Fig. 3C, Ad-TRPC3 overexpression led to
modest increases in NRVM sarcomere organization compared
with control cells infected with Ad-

-galactosidase. The posi-
tive effect of TRPC3 overexpression on sarcomere assembly
was dramatically enhanced by stimulation of cells with the
TRPC channel agonist, OAG. Enhanced sarcomere assembly
correlated with expression of ectopic TRPC3, which localized
to the perinuclear and punctuate pan-cellular regions of the
NRVMs. The perinuclear localization of TRPC3 likely repre-
sents TRPC3 transiting through the Golgi network, because
indirect immunofluorescence revealed co-localization of this
pool of TRPC3 with p155 golgin, a Golgi marker (data not
shown).
We observed that ectopic TRPC3 expression significantly
increased mean cell volume (Fig. 3D); the presence of OAG
produced a further increase. As shown in Fig. 3E, TRPC3
induced expression of ANF and
␣
-skeletal actin mRNA tran-
scripts. Of note, OAG did not appear to enhance TRPC3-me-
diated induction of these genes.
Thus, basal TRPC3 activity appears
to be sufficient to increase cell vol-
ume and induce fetal cardiac genes,
whereas full sarcomere altering
effects of TRPC3 require activation
by OAG. Together, the results sug-
gest that ectopic TRPC3 is capable
of stimulating hypertrophy of cul-
tured cardiac myocytes.
As an alternative approach to
study the role of TRPC channels in
the control of cardiac hypertrophy,
we employed two distinct small
molecule inhibitors of calcium
release-activated calcium channels,
2-APB (25) and the pyrazole deriva-
tive BTP2 (28). Whereas these
inhibitors block TRPC3-mediated
calcium influx, we cannot exclude
the possibility that 2-APB and BTP2
may also be acting on other chan-
nels including TRPVs and TRPMs.
As shown in Fig. 4, A–D, both
2-APB and BTP2 exhibited dose-
dependent inhibition of ANF secre-
tion and

-myosin heavy chain
expression in NRVMs stimulated
with PE. Only cells treated with the
highest concentrations of 2-APB
and BTP2 showed evidence of cyto-
toxicity, as determined by assessing
the presence of adenylate kinase in
culture supernatants (Fig. 4, E and
F). Treatment with 2-APB also
resulted in suppression of PE-medi-
ated increases in NRVM size and protein synthesis (data not
shown). Together, the data from these gain and loss of function
experiments suggest a role for TRPC channels in the control of
cardiac hypertrophy.
TRPC3 Activates Calcineurin/NFAT Signaling—The cal-
cineurin phosphatase plays an essential role in the regulation of
cardiac hypertrophy, and has recently been shown to be regu-
lated by TRPC3 in skeletal muscle. As such, we hypothesized
that the mechanism whereby TRPC channels stimulate cardiac
hypertrophy involves calcineurin. As an initial step to address
this hypothesis, we examined the effect of TRPC3 overexpres-
sion on the subcellular distribution of the calcineurin target,
NFAT. The NFAT transcription factor is a phosphoprotein that
resides in the cytosol and, upon dephosphorylation by cal-
cineurin, translocates to the nucleus. NFAT was cytosolic in
unstimulated NRVMs, and infection of cells with Ad-

-galac-
tosidase did not alter its subcellular distribution. In contrast,
TRPC3 overexpression partially stimulated nuclear import of
NFAT, and this effect was enhanced by treatment of the cells
with OAG (Fig. 5A). Of note, we observed modest induction of
NFAT nuclear import in Ad-

-galactosidase-infected cells
treated with OAG.
FIGURE 5. TRPC channels stimulate cardiac calcineurin/NFAT signaling. A, NRVMs were infected with
adenoviruses encoding GFP-tagged NFAT and either Ad-

-galactosidase (GAL) or Ad-TRPC3. Cells were cul-
tured in serum-free medium and exposed to OAG (100
M) for 12 h, as indicated. Cells were fixed and GFP-NFAT
localization assessed by fluorescence microscopy (top panels). TRPC3 was detected in the same cells by indirect
immunofluorescence with anti-Myc antibodies (bottom panels). B, NRVMs were treated as described in A. After 12 h
of OAG treatment, protein lysates were prepared and NFAT phosphorylation status assessed by immunoblotting
with GFP-specific antibodies (upper panel). The blot was reprobed with anti-Myc antibody to reveal ectopic TRPC3
(lower panel). C, NRVMs were left uninfected or infected with increasing amounts of Ad-TRPC3 (multiplicity of infec-
tion ⫽ 1, 5, and 25). Cells were harvested after 48 h of culture and MCIP1 expression analyzed by immunoblotting
(upper panel). Phosphorylated NFAT is indicated by circled P. The blot was reprobed to assess ectopic TRPC3 levels
(lower panel). D, NRVMs were cultured in serum-free medium and exposed to PE (20
M) in the absence or presence
of 2-APB (30
M). Following 48 h in culture, protein lysates were prepared and MCIP1 levels determined by immu-
noblotting. The 28-kDa isoform of MCIP1 is calcineurin responsive.
TRPC Channels Promote Cardiomyocyte Hypertrophy
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Immunoblotting studies were performed to confirm that
TRPC3 stimulates calcineurin-dependent dephosphorylation
of NFAT. Hypophosphorylated NFAT migrates more rapidly
than hyperphosphorylated NFAT in denaturing polyacrylam-
ide gels. As shown in Fig. 5B, overexpression of TRPC3 in
NRVMs triggered dephosphorylation of NFAT, as evidenced by
the appearance of the rapidly migrating NFAT species. Consist-
ent with the results presented in Fig. 5A, OAG enhanced
TRPC3-mediated dephosphorylation of NFAT.
To determine whether TRPC channels regulate endogenous
NFAT target genes, we monitored expression of the 28-kDa
isoform of MCIP1, which is dependent on calcineurin/NFAT
signaling. As shown in Fig. 5C, exposure of NRVMs to increas-
ing doses of Ad-TRPC3 led to selective up-regulation of 28-kDa
MCIP1,indicatingthatTRPC3overexpressionstimulatesNFAT-
dependent transcription. In contrast, 2-APB blocked PE-medi-
ated induction of 28-kDa MCIP1 (Fig. 5D). Together, the
results demonstrate that TRPC channels stimulate cardiac cal-
cineurin/NFAT signaling.
Induction of TRPC5 Expression in Failing Human Heart—To
begin to address whether TRPC channels play a role in the
pathogenesis of human heart failure, quantitative RT-PCR was
employed to survey expression of TRPC1, -3, -4, -5, and -6 in
nonfailing human heart versus hearts of patients with idio-
pathic dilated cardiomyopathy. TRPC5 mRNA expression was
significantly elevated in failing human heart relative to nonfail-
ing controls (Fig. 6A). Immunoblot analysis confirmed that
increased TRPC5 mRNA levels in failing heart correlated with
enhanced TRPC5 protein expression (Fig. 6B). We observed
two immunoreactive bands in the human heart; both were
effectively competed by preincubating the TRPC5 antibody
with antigen peptide. The lower band, of ⬃110 kDa, is in agree-
ment with the predicted molecular mass of TRPC5. Expression
of a larger specific band of ⬃180 kDa was also elevated in failing
human hearts. Consistent with previous observations (21),
TRPC3 mRNA transcripts and protein were undetectable in
nonfailing or failing human heart (Fig. 6A, and data not shown).
Levels of TRPC1, -4, and -6 were unchanged in normal versus
failing heart (data not shown).
DISCUSSION
The current study suggests that TRPC channels contribute to
the maintenance of cardiac hypertrophy via a positive feedback
mechanism involving activation of calcineurin/NFAT signaling
(Fig. 7). Pathological stress stimuli cause phospholipase C acti-
vation and store depletion, which potentiate TRPC activity and
FIGURE 6. Induction of TRPC5 protein expression in failing human heart.
A, total RNA was prepared from left ventricles of end-stage failing heart from
individuals with idiopathic dilated cardiomyopathy (n ⫽ 6) and nonfailing
controls (n ⫽ 6). Levels of mRNA transcripts for individual TRPC isoforms were
determined by quantitative RT-PCR. Values depict averages of the ratio of
TRPC mRNA to GAPDH mRNA (ng/ng), ⫾ S.D., p ⬍ 0.05. B, protein lysates were
prepared from the same LV samples and TRPC5 levels determined by immu-
noblotting (top panels). Specificity of the TRPC5 antibody was confirmed by
preincubating the antibody with the corresponding antigen peptide (middle
panels). Blots were reprobed with calnexin-specific antibody to control for
protein loading (bottom panels).
FIGURE 7. A model for regulation of cardiac hypertrophy by TRPC chan-
nels. Stress signals, such as those emanating from G protein-coupled recep-
tors (GPCRs), trigger phospholipase C (PLC) activation, with subsequent for-
mation of diacylglycerol (DAG) and inositol trisphosphate (IP3). Inositol
trisphosphate promotes release of calcium (Ca
2⫹
) stores from internal stores,
which potentiates TRPC channel activity and produces a calcium signal suffi-
cient to activate calcineurin/NFAT. TRPC channels can also be activated by
direct diacylglycerol binding. Among the genes modulated by the cal-
cineurin/NFAT pathway are TRPCs themselves, suggesting the existence of a
positive-feedback circuit that stabilizes a state of hypertrophic gene expres-
sion. NFAT regulates cardiac gene expression through association with other
transcription factors, including myocyte enhancer factor-2 (MEF2) and GATA.
TRPC Channels Promote Cardiomyocyte Hypertrophy
33494 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281• NUMBER 44 • NOVEMBER 3, 2006
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produce a calcium signal sufficient to activate calcineurin/
NFAT. Because TRPC channels are mechanosensitive, it is also
possible that abnormal mechanical stress in the hypertrophied
and failing heart might activate TRPC channels directly.
Among the genes modulated by the calcineurin/NFAT path-
way are TRPCs themselves, completing a positive feedback
circuit that stabilizes a state of hypertrophic gene expression.
Calcineurin signaling may also stabilize TRPC3 protein post-
translationally. The interplay between calcineurin and TRPC
channels may contribute to the selective activation of cardiac
calcineurin by calcium in diseased myocardium.
A similar regulatory mechanism has been hypothesized to
operate in skeletal muscle, where calcineurin/NFAT-depend-
ent up-regulation of TRPC3 provides positive feedback rein-
forcing the slow oxidative muscle fiber phenotype (22). Inter-
play between TRPC channels and calcineurin was also recently
described in a prostate cancer cell line (26). In addition, inde-
pendent pharmacologic evidence of the central role TRPC
channels play in the regulation of calcineurin/NFAT signaling
has been provided by recent small molecule screens for NFAT
inhibitors. A high-throughput screen for compounds capable
of suppressing activation of the NFAT-responsive interleu-
kin-2 promoter (27) identified a series of 3,5-bistrifluoromethyl
pyrazole store-operated calcium inhibitors (including BTP2)
(28), which have subsequently been shown to be TRPC antag-
onists (29). An alternative high-throughput screen for com-
pounds that block nuclear translocation of NFAT also pro-
duced a series of store-operated calcium inhibitors (30).
Aberrant TRPC expression or activity is thought to contrib-
ute to the pathogenesis of a number of human diseases (31).
Recent studies indicate that TRPC channels mediate the abnor-
mal calcium influx and overload observed in skeletal myotubes
from Duchenne muscular dystrophy patients (32). The patho-
logic hyperplasia of pulmonary arterial smooth muscle cells
that occurs in idiopathic pulmonary hypertension is driven, in
part, by increased calcium influx through TRPC channels (33).
Bosentan, a non-selective endothelin receptor antagonist used
for the treatment of idiopathic pulmonary arterial hyperten-
sion, may exert antiproliferative effects via down-regulation of
TRPC6 (34).
We observed that TRPC expression is up-regulated in animal
models of cardiac hypertrophy and human heart failure, and
that pharmacologic inhibition of channel activity suppressed
hypertrophy in vitro. However, the relative contribution of this
channel family to the abnormal calcium handling seen in the
hypertrophied and failing human heart remains to be further
elucidated. Intriguingly, down-regulation of cardiac SERCA2 in
vitro (a hallmark of hypertrophy in vivo) is associated with coor-
dinate up-regulation of TRPC channels and calcineurin activa-
tion, suggesting a link between the regulation of calcium
reuptake and extracellular calcium influx (35). Because TRPC
family members can form heteromultimers with distinct elec-
trophysiological properties, it will be particularly important to
assess the relative contributions of individual family members
to the activation of cardiac calcineurin/NFAT signaling. Our
own observations indicate that TRPC5, rather than TRPC3, is
up-regulated in the failing human heart, perhaps revealing a
crucial species difference. In addition, cardiac-specific overex-
pression of TRPC6 has been shown to induce heart failure in
mice.
5
As regulators of pro-hypertrophic calcium signaling in car-
diomyocytes, TRPC channels may represent novel pharmaco-
logic targets for modulating pathologic calcineurin/NFAT sig-
naling in the heart. Future work focused on the development of
selective inhibitors of TRPC channel activity or expression will
provide essential tools for the functional characterization of
these channels in cardiomyocytes, and may yield therapeuti-
cally useful compounds for the treatment of cardiac hypertro-
phy and heart failure.
Acknowledgments—We thank M. Schneider (University of Maryland)
for the NFAT-GFP adenovirus, S. McCune for SHHF rats, T. K. Pope
for rat TAB samples, M. A. McNiven (Mayo Clinic) for the antibody
against p155 golgin, and A. Tizenor for graphics. We are grateful to K.
Pitts and G. Lundgaard for assistance and advice with NRVMs, and
all members of Myogen R&D for support during the course of this
work.
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TRPC Channels Promote Cardiomyocyte Hypertrophy
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Michael R. Bristow, Eric N. Olson and Timothy A. McKinsey
Erik W. Bush, David B. Hood, Philip J. Papst, Joseph A. Chapo, Wayne Minobe,
Hypertrophy through Activation of Calcineurin Signaling
Canonical Transient Receptor Potential Channels Promote Cardiomyocyte
doi: 10.1074/jbc.M605536200 originally published online September 1, 2006
2006, 281:33487-33496.J. Biol. Chem.
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