Download Stromal interaction molecule 1 is essential for normal cardiac

Am J Physiol Heart Circ Physiol 306: H1231–H1239, 2014.
First published February 28, 2014; doi:10.1152/ajpheart.00075.2014.
Rapid Report
Stromal interaction molecule 1 is essential for normal cardiac homeostasis
through modulation of ER and mitochondrial function
Helen E. Collins,1 Lan He,2 Luyun Zou,1 Jing Qu,3 Lufang Zhou,3 Silvio H. Litovsky,1 Qinglin Yang,2
Martin E. Young,3 Richard B. Marchase,4 and John C. Chatham1
1
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama; 2Department of Nutrition Sciences,
University of Alabama at Birmingham, Birmingham, Alabama; 3Division of Cardiovascular Disease, Department of Medicine,
University of Alabama at Birmingham, Birmingham, Alabama; and 4Department of Cell, Developmental and Integrative
Biology, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 3 February 2014; accepted in final form 24 February 2014
store-operated Ca2⫹ entry; STIM1; cardiomyocytes; ER stress; mitochondria
Ca2⫹ homeostasis is critical for normal cellular function and response to stress. This is especially important
in the heart, where precise control of Ca2⫹ is essential for
excitation-contraction coupling as well as Ca2⫹-mediated signaling. In cardiomyocytes it is generally accepted that voltagedependent Ca2⫹ channels function as the predominant pathway
for Ca2⫹ entry across the sarcolemma, leading to subsequent
Ca2⫹-induced Ca2⫹ release from the endoplasmic reticulum/
sarcoplasmic reticulum (ER/SR), which is responsible for norMAINTENANCE OF
Address for reprint requests and other correspondence: J. C. Chatham, Div.
of Molecular and Cellular Pathology, Dept. of Pathology, Univ. of Alabama at
Birmingham, 1670 Univ. Blvd., Volker Hall G038, Birmingham, AL 352940019 (e-mail: [email protected]).
http://www.ajpheart.org
mal excitation-contraction coupling (1). Ca2⫹ release from the
ER/SR induced by inositol 1,4,5-trisphosphate (IP3)-generating
agonists also occurs in the heart, although its role in cardiomyocyte Ca2⫹ homeostasis still remains relatively understudied (1). However, Marchase and colleagues (18) demonstrated
that in neonatal cardiomyocytes, IP3-induced activation of
hypertrophic signaling, characterized by calcineurin-mediated
nuclear factor of activated T-cell nuclear translocation, was
dependent on an influx of extracellular Ca2⫹ that was independent of voltage-gated Ca2⫹ pathways. This type of Ca2⫹ entry,
termed store-operated Ca2⫹ entry (SOCE), was first identified
in the 1970s and is widely accepted as the primary mechanism
for Ca2⫹ signaling in nonexcitable cells. However, understanding the regulation of SOCE and its wider acceptance in excitable cells, such as cardiomyocytes, was hampered for many
years by the lack of identification of the molecular mediators of
the pathway. This changed dramatically in 2005 with the
identification of stromal interaction molecule 1 (STIM1) as the
ER/SR Ca2⫹ sensor responsible for the activation of SOCE
(33, 43).
STIM1 is a ubiquitously expressed type-I membrane protein,
with its NH2-terminus including a canonical EF-hand Ca2⫹
binding domain located in the ER/SR lumen. The COOHterminal region, which extends into the cytosol, contains a
number of key regulatory domains that play a role in facilitating SOCE (5). In the currently accepted model for STIM1mediated SOCE, a decrease in ER Ca2⫹ results in dissociation
of Ca2⫹ from STIM1, facilitating STIM1 oligomerization and
subsequent accumulation of STIM1 in ER-plasma membrane
junctions, where it interacts with Orai1 leading to Ca2⫹ entry
(21, 33). While there are several variations of this model, the
STIM1-Orai1 interaction is fundamental to the molecular
mechanism of SOCE, and the knockdown of STIM1 attenuates
SOCE (20, 22, 33, 38).
Although we and others have shown that both SOCE and
STIM1/Orai1 exist in embryonic, neonatal, and adult cardiomyocytes (18, 30, 35, 42), it should be noted that the role of
SOCE in adult cardiomyocytes remains controversial. Interestingly, while three separate groups have recently reported that
STIM1 appears to be an essential component of the pathogenesis of cardiac hypertrophy (17, 22, 38), whether this is
primarily through activation of SOCE remains to be determined. Importantly, there are also some store-independent
functions of STIM1, including regulation of arachidonic acidregulated Ca2⫹ (ARC) channels (36) and the maintenance of
ER/SR structure (11).
0363-6135/14 Copyright © 2014 the American Physiological Society
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Collins HE, He L, Zou L, Qu J, Zhou L, Litovsky SH, Yang Q,
Young ME, Marchase RB, Chatham JC. Stromal interaction molecule 1 is essential for normal cardiac homeostasis through modulation of ER and mitochondrial function. Am J Physiol Heart Circ
Physiol 306: H1231–H1239, 2014. First published February 28, 2014;
doi:10.1152/ajpheart.00075.2014.—The endoplasmic reticulum (ER)
Ca2⫹ sensor stromal interaction molecule 1 (STIM1) has been implicated as a key mediator of store-dependent and store-independent
Ca2⫹ entry pathways and maintenance of ER structure. STIM1 is
present in embryonic, neonatal, and adult cardiomyocytes and has
been strongly implicated in hypertrophic signaling; however, the
physiological role of STIM1 in the adult heart remains unknown. We,
therefore, developed a novel cardiomyocyte-restricted STIM1 knockout (crSTIM1-KO) mouse. In cardiomyocytes isolated from crSTIM1KO mice, STIM1 expression was reduced by ⬃92% with no change
in the expression of related store-operated Ca2⫹ entry proteins,
STIM2, and Orai1. Immunoblot analyses revealed that crSTIM1-KO
hearts exhibited increased ER stress from 12 wk, as indicated by
increased levels of the transcription factor C/EBP homologous protein
(CHOP), one of the terminal markers of ER stress. Transmission
electron microscopy revealed ER dilatation, mitochondrial disorganization, and increased numbers of smaller mitochondria in crSTIM1-KO hearts, which was associated with increased mitochondrial
fission. Using serial echocardiography and histological analyses, we
observed a progressive decline in cardiac function in crSTIM1-KO
mice, starting at 20 wk of age, which was associated with marked left
ventricular dilatation by 36 wk. In addition, we observed the presence
of an inflammatory infiltrate and evidence of cardiac fibrosis from 20
wk in crSTIM1-KO mice, which progressively worsened by 36 wk.
These data demonstrate for the first time that STIM1 plays an essential
role in normal cardiac function in the adult heart, which may be
important for the regulation of ER and mitochondrial function.
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STIM1 IS ESSENTIAL FOR NORMAL CARDIOMYOCYTE FUNCTION
MATERIALS AND METHODS
Materials. Unless otherwise stated, all reagents and chemicals were
obtained from Fisher Scientific.
Cardiomyocyte-restricted STIM1 knockout mouse. All animal protocols were approved by the University of Alabama at Birmingham
Institutional Animal Care and Use Committee and adhered to the
National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996). All
animals received standard chow and water on an ad libitum basis, and
lighting was maintained on a 12-h:12-h light-dark cycle. Unless stated
otherwise, the control animals used in this study refer to STIM1flox/flox/
␣-myosin heavy chain cre transgenic mice (␣-MHC-cre⫺/⫺), and these
were compared with STIM1flox/flox/␣-MHC-cre⫹/⫺ mice (crSTIM1-KO).
Data are reported for male mice only.
cr
STIM1-KO mice were generated through breeding of homozygous STIM1flox/flox mice, in which exon 2 of STIM1 was flanked by
2 lox P sites (28) with cardiomyocyte-specific ␣-MHC-cre; all mice
were on a C57/B6 background. At 21 days of age, mice were weaned,
tail-snipped, and ear-tagged, and genomic DNA was extracted from
tail snips using a Qiagen Gentra Puregene kit. PCR was performed
using genomic DNA obtained from tail snips, and as previously
described, primer pairs were used to confirm STIM1 genotype (23)
and expression of the ␣-MHC-cre transgene (26).
Adult cardiomyocytes were isolated from 20-wk control and
cr
STIM1-KO mice, as previously described (24, 25, 34).
Assessment of in vivo cardiac function. As previously described
(41), mice were initially anesthetized using 5% isoflurane-95% O2 and
restrained on a heated pad where chest hair was removed and a rectal
temperature probe was inserted to ensure maintenance of body temperature. For measurements of cardiac function, the level of isoflurane
was adjusted to 1.8%, and heart rate was continually assessed to
ensure that cardiac function was not significantly depressed. Serial
echocardiography was performed every 4 wk on adult male control
and crSTIM1-KO mice between 12 and 36 wk of age using a
Visualsonics Vevo 770 system in combination with a 30-Mhz transducer.
Tissue analyses. For histological analyses, hearts were perfused
from the apex with ice-cold PBS containing 25 mM KCl to clear
blood and to arrest the heart. Hearts were then extracted, cut into
transverse or longitudinal sections, and fixed for 24 h in 10% neutral
buffered formalin and embedded in paraffin. Sections were cut at 5
␮m and stained with hematoxylin-eosin, Masson trichrome, and picric
acid Sirius red to assess cardiac morphology, cardiac fibrosis, and
collagen deposition, respectively.
For transmission electron microscopy, LV tissue was isolated into
longitudinal sections and placed in 0.1 mol/l cacodylate buffer con-
taining 2% glutaldehyde-paraformaldehyde and was heat-fixed for 30
min to cross link proteins and aldehydes. Lipid fixation was performed
using 2% osmium tetroxide in 0.1 mol/l cacodylate buffer and 1%
aqueous uranyl acetate and tissue embedded in Epon. Transmission
electron microscopy was performed at the UAB high-resolution imaging facility, and longitudinal sections of tissue were assessed at the
level of the ER/SR membrane, mitochondria, and contractile filaments.
To determine changes in protein levels, whole heart tissue or
cardiomyocytes were initially homogenized/sonicated in lysis buffer
containing 20 mM HEPES, 1.5 mM MgCl2, 20 mM KCl, 20%
glycerol, 0.2 mM EGTA, 1% Triton X-100, 2 mM Na3VO4, 10 mM
NaF, and 2% protease inhibitor (pH 7.9). Protein was quantified using
a Bio-Rad DC assay, and lysates were prepared using 6⫻ sample
buffer, consisting of 0.5 M Tris·HCl, 10% SDS, 30% glycerol, 0.2%
␤-mercaptoethanol, and 0.012% bromophenol blue, and boiled for 5
min. Protein (25 ␮g) was separated on 7.5% or 10% SDS-PAGE gels
and subsequently transferred to polyvinylidene difluoride membranes.
Wet polyvinylidene difluoride membranes were blocked for 1 h at
room temperature with TBS-Tween 20 and 5% nonfat milk (except
for STIM2, which was incubated with PBS-casein) and incubated
overnight at 4°C with primary antibodies specific for STIM1 (1:1,000,
Cell Signaling No. 4916), STIM2 (1:1,000, Cell Signaling No. 4917),
Orai1 (1:1,000, ProSci No. 4281), mitofusin 1 (Mnf1; 1:100, Santa
Cruz Biotechnology), mitofusin 2 (Mfn2; 1:500, Abcam), dynaminrelated protein 1 (DRP-1, 1:1,000, Fisher Thermo Scientific), calsequestrin (1:5,000, Abcam No. 3516), and ER stress-specific antibodies
(Cell Signaling No. 9956) for binding immunoglobulin protein/78
kDa glucose-regulated protein (BiP/GRP78; 1:1,000) and C/EBP
homologous protein (CHOP; 1:1,000), inositol-requiring enzyme 1␣
(IRE1␣; 1:500), and protein disulfide isomerase (PDI; 1:1,000), after
which membranes were washed in TBS-Tween 20. Membranes were
incubated with the appropriate horseradish peroxidase-conjugated
secondary antibodies for 2 h at 4°C. Blots were exposed to enhanced
chemiluminescence detection, and images were obtained using autoradiograph film. Expression was normalized to the loading control
GAPDH (1:5,000, Santa Cruz Biotechnology) in whole heart tissue
and ␤-actin (1:1,000, Abcam) in cardiomyocytes.
Statistical analyses. Statistical significance was calculated using a
two-way repeated-measure ANOVA, followed by a Bonferroni post
hoc test or a Student’s t-test, as appropriate, using GraphPad Prism 4.
P values of ⬍0.05 were considered significant. Data are presented as
means ⫾ SE. All experiments comprise at least 3 to 4 mice per
experimental manipulation. Densitometry of Western blots, quantification of cardiac fibrosis and collagen deposition, and quantification
of mitochondrial size and number were performed using NIH ImageJ
software.
RESULTS
To confirm deletion of STIM1 from cardiac tissue, we
quantified STIM1 protein levels in both whole heart and
isolated adult cardiomyocytes from 20-wk-old control and
cr
STIM1-KO mice. In whole heart tissue there was approximately a 20% reduction in total STIM1 protein expression in
the crSTIM1-KO group compared with control (Fig. 1A); however, in cardiomyocytes isolated from crSTIM1-KO mice,
STIM1 was virtually undetectable with a ⬎90% reduction in
its expression (Fig. 1B). These data clearly demonstrate the
successful deletion of STIM1 in cardiomyocytes from
cr
STIM1-KO mice. The modest reduction in STIM1 in the
whole heart reinforces the notion that STIM1 protein levels are
relatively low in adult cardiomyocytes, relative to their levels
in noncardiomyocyte cells such as smooth muscle cells, endothelial cells, and fibroblasts. Furthermore, to establish whether
cardiac-specific deletion of STIM1 resulted in any compensa-
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Since STIM1 is highly conserved and appears to be ubiquitous in all eukaryotic cells (6), it seems likely that in addition
to its pathophysiological role, STIM1 may also play an important physiological role in the heart; however, to date, remarkably little is known of the physiological role of STIM1 in the
adult heart. Ubiquitous germ-line deletion of STIM1 is embryonically lethal (27); therefore, to determine the physiological
role of STIM1 in the heart, we generated a cardiomyocyterestricted STIM1 knockout (crSTIM1-KO) mouse. We found
that as early as 12 wk of age, there is evidence of ER stress and
mitochondrial abnormalities in crSTIM1-KO hearts; between
20 and 36 wk of age, crSTIM1-KO mice exhibit a progressive
decline in left ventricular (LV) function with the development
of a dilated cardiomyopathy. This study demonstrates for the
first time that STIM1 is essential for normal cardiomyocyte
homeostasis potentially through maintenance of ER/SR and
mitochondrial function.
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tory changes in other known SOCE proteins, we analyzed
STIM2 and Orai1 levels in control and crSTIM1-KO cardiomyocytes and found no differences in expression between
genotypes (Fig. 1C). In addition, analysis of STIM1 expression
in skeletal muscle revealed no differences between control and
cr
STIM1-KO groups, confirming that the changes in STIM1 are
cardiac-specific (data not shown). Since calsequestrin is responsible for buffering ER/SR Ca2⫹ levels, we examined
levels of calsequestrin in cardiomyocytes isolated from 20-wkold control and crSTIM1-KO and found no significant difference between the genotypes (Fig. 1D).
cr
STIM1-KO mice were born within the expected Mendelian
frequency and survive into adulthood with no obvious physical
differences or changes in body weight. Unbiased gene array
analyses on whole heart tissue from 12-wk-old male mice
using the Illumina mouse WG-6 2.0 expression bead chip
revealed only 15 genes that were differentially expressed
between the two genotypes, suggesting that STIM1 deletion
did not result in major adaptive/compensatory changes in the
heart (Fig. 1E). Interestingly, there were significant increases
in atrial natriuretic peptide, brain natriuretic peptide, and
␣-skeletal actin in crSTIM1-KO versus control, which are
characteristic of cardiac stress responses.
It has been proposed that one role for STIM1 is to ensure
that ER/SR Ca2⫹ levels are maintained at levels sufficient for
appropriate protein folding and processing (10, 12); consequently, the lack of STIM1 could affect ER/SR Ca2⫹ levels,
leading to increased ER stress that may promote cardiac
dysfunction. Therefore, we quantified the levels of ER chaperone proteins (IRE1␣, CHOP, BiP/GRP78, and PDI) in hearts
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Fig. 1. Stromal interaction molecule 1 (STIM1)
protein expression in whole heart tissue (A) and
isolated cardiomyocytes (B) from adult male
control and STIM1 knockout (crSTIM1-KO)
mice. C: STIM2 and Orai1 expression in cardiomyocytes from adult male control and
cr
STIM1-KO mice. D: calsequestrin protein expression in 20-wk cardiomyocytes isolated from
adult male control and crSTIM1-KO mice. Control, n ⫽ 4; crSTIM1-KO, n ⫽ 4. Representative
immunoblots (A–D: left) and mean densitometric
data (A–D: right) from 4 individual experiments
normalized to GAPDH (A) and ␤-actin (B–D)
**P ⬍ 0.01 and *P ⬍ 0.05 vs. aged-matched
control; Student’s t-test. ns, Not significant. E:
heat maps from unbiased gene expression array
data were performed using the Illumina mouse
WG-6 2.0 expression bead chip kit on hearts
from crSTIM1-KO (n ⫽ 6) and control (n ⫽ 6)
mice of 12 wk of age. Full gene name
corresponds to *IGHV1S35_M12376_Ig_
heavy_variable_1S35_13 and #IGHV1S120_
AF025443_Ig_heavy_variable_1S120_8.
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STIM1 IS ESSENTIAL FOR NORMAL CARDIOMYOCYTE FUNCTION
from 12-, 20-, and 36-wk control and crSTIM1-KO mice (Fig. 2).
Figure 2 shows that at 12 wk, there was a significant increase
in both CHOP and PDI levels in the crSTIM1-KO group. At 20
wk, CHOP and PDI remained elevated, whereas BiP/GRP78
was significantly reduced. By 36 wk, CHOP and PDI were still
increased, whereas BiP/GRP78 remained depressed and IRE1␣
was significantly increased in crSTIM1-KO versus control. We
also observed the same expression profiles for BiP, PDI, and
CHOP in cardiomyocytes isolated from 20-wk control and
cr
STIM1-KO mice (Fig. 2C). Analyses of these same proteins
in skeletal muscle show no differences between control and
cr
STIM1-KO groups, confirming that the changes in ER stress
are cardiac-specific (data not shown).
There is growing appreciation of the importance of cross talk
between ER/SR and mitochondria (8). Moreover, STIM1deficient fibroblasts are reported to have altered mitochondrial
function (14); therefore, we examined mitochondria morphology with transmission electron microscopy in 20-wk control
and crSTIM1-KO mice. In Fig. 3, we show that at 20 wk of age,
cr
STIM1-KO mice exhibit pronounced ER/SR dilatation consistent with a disruption of the close ER-mitochondria association. In addition, mitochondria from crSTIM1-KO showed
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Fig. 2. Immunoblots (A) and densitometry
analysis (B) of endoplasmic reticulum (ER)
stress proteins in 12-, 20-, and 36-wk adult
control (12, 20, and 36 wk, n ⫽ 6) and
cr
STIM1-KO heart (12 and 20 wk, n ⫽ 6; 36
wk, n ⫽7) are shown. **P ⬍ 0.01 and ***P ⬍
0.001 vs. age-matched control (ANOVA/Bonferroni post hoc test). IRE1␣, inositol-requiring enzyme 1␣; CHOP, C/EBP homologous
protein; BiP (GRP78), binding immunoglobulin protein (78 kDa glucose-regulated protein);
PDI, protein disulfide isomerase; WT, wildtype; KO, knockout. C: immunoblot (left) and
densitometry (right) of ER stress proteins in
20-wk cardiomyocytes isolated from adult
control (n ⫽ 4) and crSTIM1-KO (n ⫽ 4)
hearts.
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heterogeneity in both size and number, resulting in increased
numbers of smaller mitochondria, which were abnormally
distributed throughout the tissue. We also observed that the
mitochondria from crSTIM1-KO mice exhibited disruptions in
cristae associated with mitochondrial fragmentation and increased focal vacuolization.
In light of the changes in mitochondrial morphology in
cr
STIM1-KO hearts, we quantified the levels the mitochondrial
fission proteins FIS1 and DRP-1 and the mitochondrial fusion
proteins Mfn1, Mfn2 and optic atrophy 1. We found that there
was a significant increase in DRP-1 coupled with a significant
reduction in Mfn2 levels in 20-wk crSTIM1-KO, with no
change in Mfn1 expression (Fig. 3D). There were also no
differences in FIS1 or optic atrophy 1 between groups (data not
shown). These results are consistent with an increase in mito-
chondrial fission in crSTIM1-KO hearts and could be a contributing factor to the reduction in mitochondrial size and the
increase in mitochondrial number we observe.
To determine whether the lack of STIM1 had any adverse
effects on cardiac function, serial echocardiography was performed in crSTIM1-KO and control littermate mice every 4 wk,
from 12 to 36 wk of age (Fig. 4). From 12 to 20 wk of age,
there were no differences in cardiac function with respect to
genotype; however, as shown in Fig. 4B, from 20 to 36 wk of
age there was a progressive decline in ejection fraction in the
cr
STIM1-KO mice. Representative M-mode echo images at 12
and 36 wk as shown in Fig. 4A highlight the marked LV
dilatation in the crSTIM1-KO mice at 36 wk, which is supported by the increase in LV volume (Fig. 4C) and decrease in
ejection fraction (Fig. 4B). Although there was also a signifi-
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Fig. 3. Transmission electron micrograph
images taken from left ventricular (LV) tissue isolated from 20-wk-old control (A) and
cr
STIM1-KO (B) mice. Scale bar equals 500
nm, and red arrows highlight ER dilatation.
C: mean measurements of mitochondrial
length and number of mitochondria per 750
␮m2 grid taken at 1,100⫻ magnification
from 20-wk crSTIM1-KO and control mice.
Measurements were taken using ImageJ
from at least 10 different images per 3 different hearts. **P ⬍ 0.001 and ***P ⬍
0.0001, Student’s t-test. D: Western blot and
densitometry analysis of mitochondrial fusion proteins Mfn1 and Mfn2 and the mitochondrial fission protein dynamin-related
protein 1 (DRP-1) in 20-wk control and
cr
STIM1-KO heart. *P ⬍ 0.05 vs. control
(Student’s t-test). Control, n ⫽ 6; crSTIM1KO, n ⫽ 6.
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cant reduction in LV wall thickness between control and
cr
STIM1-KO, this difference was due to a significant increase
in LV wall thickness in the controls between 20 and 36 wk
(Fig. 4D), possibly reflecting a lack of active cardiac growth in
the crSTIM1-KO. In addition, there were no differences in LV
mass or in heart weight-to-tibia length ratio between groups
(data not shown). The increase in LV volume and difference in
wall thickness in crSTIM1-KO mice was also evident in tissue
sections from 36-wk animals (Fig. 4E), characteristic of the
presence of a dilated cardiomyopathy. High-magnification
analysis of histological sections revealed the presence of an
inflammatory infiltrate and cardiac fibrosis in crSTIM1-KO at
20 wk, which progressively worsened by 36 wk and was not
present in control hearts (Fig. 4F). Kaplan-Meier survival
curve analysis (Fig. 4G) demonstrates that crSTIM1-KO mice
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Fig. 4. Serial echocardiography was performed from 12 to 36 wk of age for quantification of cardiac function and histology in
control and crSTIM1-KO mice. A: representative M-mode echocardiography traces taken
from 12 and 36 wk control and crSTIM1-KO.
B: ejection fraction (in %) as a function of
age. C and D: diastolic LV volume (C) and
LV posterior wall thickness at diastole (LVPWd)
at 20 and 36 wk of age. *P ⬍ 0.05 and
**P ⬍ 0.001 vs. age-matched control
(ANOVA/Bonferroni post hoc test). E:
transverse whole heart images of hematoxylin and eosin (H&E) staining from 20- and
36-wk-old control and crSTIM1-KO, taken
at 1.25⫻. Scale bar represents 2 mm. F:
H&E, Masson trichrome, and picric acid
Sirius red stained sections of hearts from 20and 36-wk-old control and crSTIM1-KO.
Images taken at 40⫻, and scale bar equals 50
␮m. Control (20 wk, n ⫽ 6; and 36 wk, n ⫽
7) crSTIM1-KO (20 wk, n ⫽ 14; and 36 wk,
n ⫽ 8). G: Kaplan-Meier survival curve of
cr
STIM1-KO vs. age-matched control mice
(␹2, P ⫽ 0.0001, n ⫽ 20 per genotype).
begin to die at 40 wk of age and that over 60% of crSTIM1-KO
die before 50 wk of age versus control mice, which exhibit no
death throughout the duration of this time course (P ⬍ 0.0001).
It has been reported that ␣-MHC-cre mice exhibit agedependent decline in cardiac function (4); therefore, we performed serial echos on a cohort of ␣-MHC-cre and wild-type
mice. Using two-way repeated-measure ANOVA, unlike the
differences observed between crSTIM1-KO and control mice,
we found by 36 wk of age there were no significant differences
in ejection fraction, LV volume, or LV wall thickness between
␣-MHC-cre and wild-type mice (data not shown). In addition,
we have found that at 12 wk, unlike our crSTIM1-KO mice,
there was no significant difference in the expression of ER
stress markers between ␣-MHC-cre and wild-type mice
(data not shown), confirming that induction of ER stress is
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STIM1 IS ESSENTIAL FOR NORMAL CARDIOMYOCYTE FUNCTION
cr
STIM1-KO specific. We did not observe any additional
adverse effects of cre-recombinase in the ␣-MHC-cre mice;
moreover, unlike the crSTIM1-KO mice, we did not observe a
dramatic decrease in survival in ␣-MHC-cre between 40 and
50 wk of age (data not shown).
DISCUSSION
there were increased inflammatory infiltrate and cardiac fibrosis in crSTIM1-KO hearts, indicative of additional cardiomyocyte stress and injury. While much of the focus on the role of
STIM1 as a key mediator of SOCE and Ca2⫹ signaling, it has
also been suggested that STIM1 may play a critical role in
regulating ER/SR function to ensure appropriate folding and
processing of proteins (37). This is supported by the fact that as
early as 12 wk of age, there is a significant elevation in CHOP
and PDI proteins in crSTIM1-KO hearts, which persists up to
36 wk of age. Interestingly, BiP/GRP78 protein is normal at 12
wk but is markedly reduced at 20 wk and remains depressed
through 36 wk. The reduction in BiP/GRP78 protein levels is
somewhat surprising since it is typically increased in response
to acute ER stress and is responsible for binding to misfolded
proteins to prevent aggregation, as well as the activation of
CHOP. The lack of STIM1, however, likely represents a more
chronic or sustained ER stress, and as such additional studies
are required to understand the mechanism(s) contributing to the
lower levels of BiP/GRP78. Nevertheless, these results are
consistent with other reports indicating that STIM1 may mediate ER remodeling (11), as well as associate with several
additional ER chaperone proteins such as stanniocalcin 2 (42)
and the ER oxidoreductase ERp57 (32).
There is increasing evidence to show that there is a close
association between ER/SR and mitochondria (8, 40) and that
the progression of ER stress can lead to impaired mitochondrial
function (2, 3). Interestingly, Henke et al. (14) reported that
mouse embryonic fibroblasts lacking STIM1 exhibited abnormal mitochondrial function and morphology. These observations are consistent with our observations of altered mitochondrial morphology and disorganization in crSTIM1-KO hearts.
Moreover, the changes in Mfn2 and DRP-1 seen here are
indicative of a shift toward mitochondrial fission, which could
account for the decrease in mitochondrial size and increased
number of mitochondria observed in the crSTIM1-KO hearts. It
should be noted that Mfn2 rather than Mfn1 has been shown to
play a key role in mitochondrial ER/SR tethering (7) and may
also regulate trafficking of STIM1 to the plasma membrane
(35); consequently, the decrease in Mfn2 in the crSTIM1-KO
hearts could be a contributing factor to the changes in mitochondrial morphology. While the mechanism underlying the
decrease in Mfn2 in response to the loss of STIM1 remains to
be determined, this observation underscores earlier reports
indicating an interrelationship between Mfn2 and STIM1.
Cardiomyocytes are regularly exposed to an extracellular
neurohormonal milleu, which includes the IP3-generating agonists, such as angiotensin II and endothelin, both of which are
known to stimulate SOCE (29). It is possible, therefore, that in
the setting of the crSTIM1-KO heart that the adverse effects we
observe are due in part to the activation of IP3 receptor (IP3R)
in the ER/SR in the absence of functional STIM1. If this indeed
the case, then continued activation of IP3R could result in a
prolonged reduction in ER/SR Ca2⫹ levels, normally sensed by
STIM1, which may contribute to the observed induction of ER
stress we observe since a reduction in ER/SR Ca2⫹ is known to
induce ER stress (10, 12). It should also be noted that in
addition to SOCE, STIM1 has been reported to regulate arachidonate-regulated Ca2⫹ (ARC) channels a store-independent
Ca2⫹ entry pathway (36); consequently, a lack of STIM1 may
also impair this Ca2⫹ entry pathway. However, since the
function of ARC channels in the heart remains unclear, future
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STIM1 was first identified in 1996 as a single-pass ER
membrane protein with unknown function; however, in 2005,
it was recognized as a required component of SOCE, and since
then its role in regulating SOCE has been extensively examined, particularly in nonexcitable cells. It is also increasingly
apparent that STIM1 has functions that are independent of
SOCE, including regulation of ARC channels (36) and maintenance of ER/SR function (11). While there has been a
growing body of evidence demonstrating that STIM1 is present
in cardiomyocytes (19, 22, 37, 44), particularly as a mediator
of cardiac hypertrophy (13, 17, 22, 38), the physiological role
of STIM1 in the adult heart remains poorly understood. We
have demonstrated for the first time that STIM1 plays an
essential role in maintaining cardiomyocyte homeostasis. Specifically, we have shown that a ⬎90% reduction in STIM1
expression in cardiomyocytes resulted in increased ER stress
and changes in mitochondrial morphology consistent with
increased mitochondrial fission that were associated with increased mortality, impaired LV function, and the development
of a dilated cardiomyopathy.
The primary focus of STIM1 studies in the heart have been
on the role of STIM1 in regulating hypertrophic signaling;
however, the fact that STIM1 is evolutionarily highly conserved and is reported to be present in all eukaryotic cells (6)
strongly suggests that it also plays an important homeostatic
role. Earlier studies had reported that cardiomyocyte STIM1
protein levels decline from the embryonic to the adult heart,
only to increase in response to pressure overload (17, 22).
These observations raised the possibility that STIM1 might
play an important role in early development of the heart and
that like many stress-related proteins, it reemerges in the adult
heart subjected to increased hemodynamic stress. However, if
this were indeed the case, it would be anticipated that
cr
STIM1-KO would either be embryonically lethal or lead to
severe phenotype in young animals. It is clear that this is not
the case since crSTIM1-KO mice are born with the normal
Mendelian frequency and survive into young adulthood with
no overt phenotype. Indeed, the fact that there are no gross
morphological or functional differences between crSTIM1-KO
and control hearts up to 20 wk of age indicates that STIM1 is
likely unnecessary for normal cardiac development and function. On the other hand, the rapid development of a dilated
cardiomyopathy between 20 and 36 wk and significant mortality starting around 40 wk of age in the crSTIM1-KO group
strongly suggests that STIM1 is required for maintaining normal cardiomyocyte homeostasis in the adult heart.
One indication of the potential importance of STIM1 in adult
cardiomyocytes was evident in the gene array analyses where
despite relatively minor differences between crSTIM1-KO and
control groups, there were significant increases in brain natriuretic peptide and atrial natriuretic peptide, indicative of an
early stress response. Furthermore, at 20 wk of age, even
though functionally there were no differences between groups,
H1237
Rapid Report
H1238
STIM1 IS ESSENTIAL FOR NORMAL CARDIOMYOCYTE FUNCTION
ACKNOWLEDGMENTS
We acknowledge Drs. Anjana Rao and Masatsugu Oh-hora for the kind
donation of STIM1flox/flox mice; our animal care staff Casie Carson and Lisa
Unangst for excellent animal care, Ed Phillips and Melissa Foley-Chimento for
assistance with transmission electron microscopy; David Crossman for discussions on gene ontology and all members of the Yang, Young, and Chatham
Laboratories for technical assistance and valuable discussions.
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grant R21-HL-110366 (to J. C. Chatham); William W Featheringill Postdoctoral Fellowship from the UAB Comprehensive Cardiovascular Centre (to
H. E. Collins); and the Small Animal Physiology Core of the UAB Diabetes
Research Center supported by NIH Grant 2P30-DK-079626.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
H.E.C., Q.Y., M.E.Y., and J.C.C. conception and design of research;
H.E.C., L.H., L. Zou, J.Q., and L. Zhou performed experiments; H.E.C. and
S.H.L. analyzed data; H.E.C., L. Zhou, S.H.L., M.E.Y., R.B.M., and J.C.C.
interpreted results of experiments; H.E.C. prepared figures; H.E.C. drafted
manuscript; H.E.C., L. Zhou, S.H.L., Q.Y., M.E.Y., R.B.M., and J.C.C. edited
and revised manuscript; H.E.C., L. Zhou, S.H.L., Q.Y., M.E.Y., R.B.M., and
J.C.C. approved final version of manuscript.
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cr
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addition, the fact that there was no evidence of increased ER
stress in 12 wk old in hearts from ␣-MHC-cre mice further
supports the fact that these effects are specific for the lack of
STIM1. However, this could be further confirmed using
inducible, cardiomyocyte-specific STIM1-KO models, alternatively small interfering RNA approaches to chronically
knockdown STIM1 as described by Hulot et al. (17) could
be particularly valuable in this regard as they avoid the
complications that can be associated with the different
inducible KO models.
In conclusion, we have shown that the absence of STIM1 in
cardiomyocytes is associated with ER stress as early as 12 wk
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fission and fusion and ultimately the development of a dilated
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