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Introduction
In response to mechanical and neurohumoral stimuli, cardiac muscle undergoes adaptive hypertrophic growth,
characterized by increases in myocyte size, protein synthesis and re-expression of fetal cardiac genes in order to maintain cardiac
output. Although initially beneficial, sustained cardiac hypertrophy can be deleterious because of increased
risk for the development of heart failure and lethal
arrhythmias[1,2].
It has been demonstrated in vivo and in vitro
that, by activating phospholipase C (PLC), Gq protein-coupled receptors
(GqCRs) relay the signals of mechanical overload and neurohumoral factors, such as catecholamine, angiotensin-II and
endothelin-1, to initiating cellular hypertrophic
response[3,4]. PLC catalyzes the cleavage of
polyphosphoino-sitide into dual signaling molecules, inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol (DAG), which, in turn, activate downstream protein
kinases and phosphatases, such as Ca2+/calmodulin (CaM)-dependent
calcineurin[5,6] and CaM kinases
(CaMKs)[7,8], and protein kinase C
(PKC)[9]. These signaling pathways then act independently or crosstalk with each other, converging
on the cellular hypertrophic
responses[2,9,10]. Of these, elevation in intracellular
Ca2+ concentration is implicated as an important
factor to initiate and perpetuate GqCR-mediated cardiac hypertrophy via CaM/carcineurin- and CaMK-mediated activation of
transcription factors, such as nuclear factor of activated T-cells (NFAT) and myocyte enhancer factor-2
(MEF2)[5_9]. Understanding the regulation of intracellular
Ca2+ signaling by the
PLC-IP3-IP3 receptor
(IP3R) pathway is thus crucial to understanding cellular mechanisms responsible for GqCR-mediated cardiac
hypertrophy.
In the heart, the main source of
Ca2+ in excitation-contraction is controlled by ryanodine receptors (RyRs), whereas the
role of IP3Rs, which express much less than RyRs, remained
obscure[11]. Recently, however,
IP3Rs have been found to be abundantly expressed in the embryonic
heart[12,13] and in the adult heart under conditions of heart failure and chronic
arrhythmias[14,15]. This implicates the roles of
IP3R-mediated Ca2+ signaling in cardiac development or remodeling in response to stress.
Therefore, the present study was aimed to determine
IP3R regulation of intracellular
Ca2+ signaling. In particular, we intended
to delineate possible roles for IP3R
Ca2+ signaling in hypertrophic growth induced by sustained stimulation of a prototypical
GqCR, a1 adrenergic receptor
(a1AR), in ventricular myocytes from the developing heart.
Materials and methods
Isolation and culture of myocytes Neonatal rat ventricular myocytes (NRVMs) were isolated from 1 to 2-d-old
Sprague-Dawley rats by enzymatic digestion with 0.1% trypsin (Hyclone) and 0.03% collagenase (Worthington Biochemical), as
described in a previous study[16].
After incubating the cell-containing supernatant for 1.5 h to remove fibroblasts, cells were
plated onto laminin-treated 35-mm dishes at a density of
1.0×103-1.2×103
cells/mm2 unless specified otherwise. The culture
medium, Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) and Medium 199 (4:1) containing 10% fetal bovine serum, 4
mmol·L-1 L-glutamine, 100 U/mL penicillin and streptomycin, and 0.1
mmol·L-1 5-bromo-2-deoxyuridine (Roche Molecular Biochemicals),
was replaced at 42 h with serum-free medium, and cells were further cultured for 6 h. Cultured cells
exhibited >95% positive staining for
a-actinin, and began spontaneous contraction 24_48 h into
culture.
Reverse transcription-polymerase chain
reaction Myoc ytes were lysed with TRIzol reagent (Invitrogen) and clarified
by centrifugation. Analysis of mRNA levels for atrial natriuretic factor (ANF) and 18 s were performed with primers designed
to detect rat gene products. ANF detection used primers 5¡¯-TCCCAGGCCATATTGGAGCA-3¡¯ and
5¡¯-CAGCGAGAGCCCTCAGT-3¡¯, generating a 306 bp fragm ent. Detection of mRNA 18 s used primers
5¡¯-TGCAGCCCCGGA-CATCTAAG-3¡¯ and 5¡¯-GGAAGGGCACCACCAGGAGT-3¡¯, generating a 317 bp fragment.
RT-PCR reactions were performed with 2 µg of total RNA and followed by
30 cycles of PCR amplification.
Confocal Ca2+ imaging NRVMs were loaded with 4
µmol/L Fluo-4/AM (Molecular Probes) in culture medium at 37
oC for 30 min and then were washed with HEPES-buffered salt
solution (mmol·L-1: NaCl 135, KCl 5,
MgCl2 1, CaCl2 1.8,
HEPES 10 and glucose 11, with pH 7.4 adjusted by NaOH) for 20 min. Confocal images of Fluo-4 fluorescence (excitation at
488 nm and emission detection at >515 nm) were obtained using Leica SP2 inverted microscope equipped with a 63×oil
immersion objective (NA 1.4). Time-lapsed
(xy, 1.63 s/frame) or linescan (xt, 2 ms/line, 0.15 µm/pixel) images were obtained
with 1.5-µm axial resolution. Image data analysis used customer-devised routines coded in the Interactive Data Language
(IDL, Research System). All experiments were performed at room temperature (22_24
oC).
Assay of cellular hypertrophy After 6-h culture in serum-free DMEM, NRVMs at a density of
1×103/mm2 were stimulated
with 10 µmol·L-1 phenylephrine (PE), a selective
a1AR agonist, for 48 h to induce cell hypertrophy. In experiments with
pharmacological pretreatment, designated reagents were applied 10_30 min prior to PE application and then kept in culture
medium. Cell areas were measured by planimetry with
3-5 frames/dish captured at ×40 magnification (Leica software) for >100
cells in each treatment. Relative rates of protein synthesis were
determined by incubation of myocytes with 2
µCi·mL-1 [3H]
leucine (Amersham, UK) for 6 h and then incubated in 10% trichloroacetic acid for 30 min on ice to precipitate the protein. The
precipitates were washed twice with cold water and then were solubilized in 0.1% SDS+0.3
mol·L-1 NaOH at 37 oC for 1 h.
[3H]leucine incorporation was then quantified for radioactivity (Beckman LS 1801) from triplicate aliquots of each
sample.
Materials 2-Aminoethoxydiphenylborate (2-APB) and xestospongin C (Xe C) were from Calbiochem. Ryanodine and
phenylephrine (PE) were purchased from Sigma-Aldrich. Myo-inositol 1,4,5-trisphosphate hexakis (butyryloxymethyl) ester
(IP3BM) was synthesized as described in a previous study (purity
>95%)[17].
Statistical analysis The data were analyzed and presented as mean±SEM. When appropriate, statistical comparison
was carried out with two-way paired or unpaired Student¡¯s
t-test. The accepted level of significance was
P<0.05.
Results
IP3BM-induced potentiation of
Ca2+ oscillations In a previous study (submitted to PNAS) we identified that
IP3 formation and spontaneous global
Ca2+ transients were increased in a dose-dependent manner after
a1AR stimulation with PE. Additionally, local
Ca2+ sparks and waves, especially in the perinuclear area, were enhanced significantly upon PE stimulation,
concomitant with the enriched distribution of
IP3Rs in this area. More importantly, inhibition of
IP3Rs with membrane permeable
IP3R inhibitors, 2-APB and Xe C, significantly inhibited these PE effects, suggesting an involvement of
IP3R activation for the PE-enhanced
Ca2+ signaling in the developing
cardiomyocytes.
To confirm this hypothesis, we applied
IP3BM, a membrane permeant ester of
IP3 to activate IP3Rs
directly[17], bypassing a1AR. First, we used a type of non-excitable cell, HEK293 cells, that dominantly express
IP3Rs as the intracellular
Ca2+ release channels to identify specific properties of this synthesized
compound[18]. As
reported[17], exposure of
IP3BM (2_25
µmol·L-1) for 5 min induced dose-dependent intracellular
Ca2+ release in a Ca2+-free medium, with approximately 70% of maximal
Ca2+ release seen at a concentration of 10
µmol·L-1 (data not shown). Thus,
IP3BM (10 µmol·L-1) stimulating NRVMs continuously
for 6 min was used throughout the following study. Figure 1A shows that the frequency of spontaneous
Ca2+ oscillations in minolayer NRVMs was increased in the presence of
IP3BM, from 5.40±0.36
min-1 in control (n=12) to 14.50±1.38
min-1 (n=7, P<0.01 vs
control). Pretreatment of cells with 2-APB 4
µmol·L-1 or Xe C 10
µmol·L-1 for 10 min robustly inhibited
IP3BM-induced potentiation of
Ca2+ oscillations, whereas ryanodine, a RyRs inhibitor, at a concentration of 30
µmol·L-1 failed to block the
IP3BM effect (Figures 1B-D). These findings, therefore, provide direct evidence for the
IP3R signaling pathway involved in
a1AR potentiation of Ca2+ oscillations.
Subcellular responses of Ca2+ sparks to
IP3BM stimulation Ca2+ sparks are thought to constitute the elementary events
of Ca2+ waves and global
Ca2+ transients[19]. Stimulation of NRVMs with PE (10
µmol·L-1) could increase the spark production
by 163% and 86.3% in the perinuclear and cytosol regions, respectively. To further identify the underlying molecular
mechanism, we then tested this local
Ca2+ response to IP3BM using high-resolution linescan measurement across the nucleus
of a single NRVM. We defined "perinuclear sparks" as sparks whose centers were within the nucleus and its 1-µm flanking
regions seen in linescan images (Figure 2A); the rest were referred to as "cytosolic sparks".
In IP3BM-unstimulated myocytes, cytosolic sparks occurred at a space-time density of 1.15±0.11 (100
µm·s)-1, and perinuclear sparks displayed a 51% higher density (1.74±0.12
(100 µm·s)-1, n=32 cells,
P<0.05 vs cytosolic events). Similar to PE,
IP3BM increased spark frequency by 112% and 62% in the perinuclear and cytosol regions, respectively, rendering 1.8-fold
higher perinuclear than cytosol spark density (Figure 2A, 2B), while the amplitude, width and duration of
Ca2+ sparks were not significantly altered in either area. Concomi-tantly, the
IP3R inhibitor, 2-APB also completely reversed the
IP3BM-induced spark responses in both subcellular areas (Figure 2B). Therefore, these results further confirm that
a1AR stimulation profoundly modulates the genesis of
Ca2+ sparks through IP3R-dependent mechanism, and the spark response is greater in
the perinuclear region of NRVM.
IP3R inhibition suppressed PE-induced hypertrophic
responses Finally, we sought to test the involvement of the
IP3R signaling pathway in a1AR stimulated NRVM growth, a well-established model for cardiomyocyte
hypertrophy[2,6]. As previously
reported[6], NRVMs with sustained (48 h) PE stimulation manifested increased cellular size (Figure 3A, 3B)
and overall protein synthesis assayed by [3
H]leucine incorporation (Figure 3C). Additionally, mRNA expression of fetal cardiac
genes such as ANF was also elevated upon PE stimulation (Figure 3D), a hallmark of cardiac
hypertrophy[20]. Inhibition of
IP3Rs with 2-APB or Xe C, at concentrations that effectively suppressed the aforementioned
Ca2+ responses, caused a substantial suppression of PE-induced increases in cellular size, protein synthesis, and ANF mRNA level (Figure 3). Parallel
experiments confirmed that PKC inhibitor, chelerythrine, also partially reversed PE-induced hypertrophic responses (Figure
3)[9], suggesting that neither PKC nor
IP3R pathway is the exclusive mediator of the
a1AR hypertrophic effects. Indeed, combined
IP3R and PKC inhibition appeared to exert greater effects in suppressing PE-stimulated protein synthesis (Figure
3C) and ANF expression (Figure 3D). Thus, the present and previous findings support the notion that both
IP3R and PKC pathways contribute to
a1AR-mediated hypertrophic
effects[6,9].
To obtain more direct evidence for the role of
IP3R in initiating cardiac hypertrophic
growth, we also tried IP3BM (10
IP3,
µmol·L-1) in parallel experiments, but we failed to induce significant cardiomyocyte hypertrophic growth by this compound,
this is likely because of its quick metabolism of
IP3 inside the cells.
Discussion
The present study provides direct evidence for
IP3R activation as a cellular mechanism underlying
a1AR stimulated Ca2+ signaling and, more importantly, linking to hypertrophic growth in ventricular myocytes from the developing rat heart.
Hierarchical Ca2+ events in NRVMs, ranging from local
Ca2+ sparks, waves to global
Ca2+ transients, manifest a rather complex
architecture of intracellular Ca2+ signaling. Unlike
IP3BM can enter into cells and release free
IP3 inside, enabling us to observe
Ca2+ signaling response to
IP3R activation in intact cells. At the cellular level,
IP3BM stimulation increases the frequency of spontaneous
Ca2+ transients to 3-fold (Figure
1). At the elementary release level, the action of
IP3BM is characterized by an increase in the rate of spark occurrence, with
unitary properties remaining stereotypical. The spark response is spatially uneven: the perinuclear sarcoplasmic reticulum
manifests a greater increase in spark production (Figure 2), concomitant with the subcellular distribution of
IP3Rs in NRVMs (data not shown here). These similar responses in local as well as global
Ca2+ signaling to IP3BM and
a1AR stimulation are all sensitive to inhibition of
IP3Rs, further confirming activation of
IP3Rs as the key mechanism in
a1AR mediated Ca2+ signaling.
Intracellular Ca2+, particularly nuclear
Ca2+, plays an important role in the regulation of gene expression that underlies
cardiac remodeling during stress
responses[1,4,5,21]. Accumulating evidence has indicated that the enhancement of
Ca2+ signaling activates multiple downstream signaling factors that induce cardiac hypertrophic growth. For instance, an increase
of Ca2+ in cardiomyocytes activates
Ca2+-dependent phosphatase calcineurin, resulting in dephosphorylates
of transcription factors known as NFAT3, which, in turn, binds upon dephosphorylation to the
transcription factor GATA4 to upregulate
cardiac-specific gene
expression[5,6,22]. However, Ca2+
signaling also activates CaMK, which then stimulate several
transcription factors, including MEF2, and initiates hypertrophic gene
expressions[7,8]. More recently,
IP3Rs have been found to be confined to the nuclear envelope of adult rat ventricular myocytes, and associated with the activation of local
CaMK II[23], implying
GqCR-IP3R-Ca2+-CaMK II may converge to a signaling pathway at the site of the nucleus.
The observation that PE (data not shown) and
IP3BM similarly and profoundly alter
Ca2+ signaling, including sparks, waves and
Ca2+ transient frequency, and the fact that these alterations occur preferentially in the perinuclear region implicate
a role for IP3Rs in a1AR-stimulated NRVM growth. While previous studies have linked the hypertrophic responses to the
DAG-PKC branch of the a1AR signaling
cascades[9], a major finding of the present work is that
IP3R inhibitors, at concentrations that effectively block
a1AR- and IP3BM-induced
Ca2+ responses, significantly attenuate ANF expression, protein
synthesis and cell growth induced by sustained (48 h) PE stimulation. Furthermore, we demonstrated that the hypertrophic
effect of a1AR stimulation depended on the concurrent
IP3-IP3R pathway and DAG-PKC pathway (Figure 3). Either inhibition
partially reverses the hypertrophic growth while inhibition of dual pathways exerts greater inhibitory effects, thus reconciling
our new finding with those in the published
literature[24,25].
In summary, we have demonstrated that
IP3R activation plays an important role in triggering local as well as global
Ca2+ release in a1AR stimulated NRVM, and the altered intracellular
Ca2+ signaling is in part responsible for
catecholamine-induced cardiomyocyte hypertrophic growth.
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