Role of Inositol 1,4,5-Trisphosphate Receptors in Α1-Adrenergic
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Acta Pharmacologica Sinica 2006 Jul; 27 (7): 895–900 Full-length article α Role of inositol 1,4,5-trisphosphate receptors in 1-adrenergic receptor- induced cardiomyocyte hypertrophy1 Da-li LUO2,3,5, Jian GAO2, Xiao-mei LAN3, Gang WANG4, Sheng WEI4, Rui-ping XIAO4, Qi-de HAN3 2Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing 100069, China; 3Institute of Cardiovascular Science at Health Science Center, Peking University, Beijing 100083, China; 4Institute of Molecular Medicine and College of Life Science, Peking University, Beijing 100871, China Key words Abstract inositol 1,4,5-trisphosphate receptors; Ca2+ Aim: Intracellular Ca2+ plays pivotal roles in diverse cellular functions, including sparks; α adrenergic stimulation; cardiac 1 gene transcription that underlies cardiac remodeling during stress responses. hypertrophy However, the role of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the media- tion of cardiac intracellular Ca2+ and hypertrophic growth remains elusive. Prior 1 Project supported by the National Natural work with neonatal rat ventricular myocytes suggests that activation of IP Rs Science Foundation of China (No 30470692). 3 α α 2+ 5 Correspondence to Dr Da-li LUO. may be linked to 1 adrenergic receptor ( 1AR) increased stereotyped Ca spark Phn 86-10-8391-1519. occurrence and global Ca2+ oscillations. Thus, we hypothesized that Ca2+ release Fax 86-10-8391-1520. through IP Rs was necessary for α AR-stimulated cardiac hypertrophy. Methods: E-mail [email protected] 3 1 We used myoinositol 1,4,5-trisphosphate hexakis (butyryloxymethyl) ester (IP3BM), Received 2006-04-18 a membrane-permeant ester of IP3, to activate IP3Rs directly, and Fluo 4/AM to Accepted 2006-05-17 2+ µ -1 measure intracellular Ca signaling. Results: IP3BM (10 mol·L ) mimicked the α 2+ doi: 10.1111/j.1745-7254.2006.00382.x effects of phenylephrine, a selective agonist of 1AR, in increments in local Ca spark release (especially in the perinuclear area) and global Ca2+ transient frequencies. More importantly, IP3R inhibitors, 2-aminoethoxydiphenyl borate 2+ and Xestospongin C, abolished the IP3BM-induced Ca responses, and signifi- α cantly suppressed 1AR-induced cardiomyocyte hypertrophy assayed by cell size, [3H] leucine incorporation and atrial natriuretic factor gene expression, dur- ing sustained (48 h) phenylephrine stimulation. Conclusion: These results, α therefore, provide cellular mechanisms that link IP3R signaling to 1AR-stimulated gene expression and cardiomyocyte hypertrophy. response[3,4]. PLC catalyzes the cleavage of polyphosphoino- Introduction sitide into dual signaling molecules, inositol 1,4,5- In response to mechanical and neurohumoral stimuli, car- trisphosphate (IP3) and diacylglycerol (DAG), which, in turn, diac muscle undergoes adaptive hypertrophic growth, char- activate downstream protein kinases and phosphatases, such acterized by increases in myocyte size, protein synthesis as Ca2+/calmodulin (CaM)-dependent calcineurin[5,6] and CaM and re-expression of fetal cardiac genes in order to maintain kinases (CaMKs)[7,8], and protein kinase C (PKC)[9]. These cardiac output. Although initially beneficial, sustained car- signaling pathways then act independently or crosstalk with diac hypertrophy can be deleterious because of increased each other, converging on the cellular hypertrophic re- risk for the development of heart failure and lethal arrhythmias[1,2]. sponses[2,9,10]. Of these, elevation in intracellular Ca2+ con- It has been demonstrated in vivo and in vitro that, by centration is implicated as an important factor to initiate and activating phospholipase C (PLC), Gq protein-coupled re- perpetuate GqCR-mediated cardiac hypertrophy via CaM/ ceptors (GqCRs) relay the signals of mechanical overload carcineurin- and CaMK-mediated activation of transcription and neurohumoral factors, such as catecholamine, angio- factors, such as nuclear factor of activated T-cells (NFAT) tensin-II and endothelin-1, to initiating cellular hypertrophic and myocyte enhancer factor-2 (MEF2)[5–9]. Understanding ©2006 CPS and SIMM 895 Luo DL et al Acta Pharmacologica Sinica ISSN 1671-4083 2+ the regulation of intracellular Ca signaling by the PLC-IP3- PCRamplification. 2+ IP3 receptor (IP3R) pathway is thus crucial to understanding Confocal Ca imaging NRVMs were loaded with 4 µmol/L cellular mechanisms responsible for GqCR-mediated cardiac Fluo-4/AM (Molecular Probes) in culture medium at 37 oC for hypertrophy. 30 min and then were washed with HEPES-buffered salt 2+ -1 In the heart, the main source of Ca in excitation-con- solution (mmol·L : NaCl 135, KCl 5, MgCl2 1, CaCl2 1.8, traction is controlled by ryanodine receptors (RyRs), whereas HEPES 10 and glucose 11, with pH 7.4 adjusted by NaOH) the role of IP3Rs, which express much less than RyRs, re- for 20 min. Confocal images of Fluo-4 fluorescence (excitation [11] mained obscure . Recently, however, IP3Rs have been at 488 nm and emission detection at >515 nm) were obtained found to be abundantly expressed in the embryonic heart[12,13] using Leica SP2 inverted microscope equipped with a 63×oil and in the adult heart under conditions of heart failure and immersion objective (NA 1.4). Time-lapsed (xy, 1.63 s/frame) [14,15] chronic arrhythmias . This implicates the roles of IP3R- or linescan (xt, 2 ms/line, 0.15 µm/pixel) images were obtained mediated Ca2+ signaling in cardiac development or remodel- with 1.5-µm axial resolution. Image data analysis used cus- ing in response to stress. Therefore, the present study was tomer-devised routines coded in the Interactive Data Lan- 2+ aimed to determine IP3R regulation of intracellular Ca guage (IDL, Research System). All experiments were per- signaling. In particular, we intended to delineate possible formed at room temperature (22–24 oC). 2+ roles for IP3R Ca signaling in hypertrophic growth induced Assay of cellular hypertrophy After 6-h culture in se- α 3 2 by sustained stimulation of a prototypical GqCR, 1 adrener- rum-free DMEM, NRVMs at a density of 1×10 /mm were α -1 gic receptor ( 1AR), in ventricular myocytes from the devel- stimulated with 10 µmol·L phenylephrine (PE), a selective α oping heart. 1AR agonist, for 48 h to induce cell hypertrophy. In experi- ments with pharmacological pretreatment, designated re- Materials and methods agents were applied 10–30 min prior to PE application and then kept in culture medium. Cell areas were measured by Isolation and culture of myocytes Neonatal rat ventricu- planimetry with 3−5 frames/dish captured at ×40 magnifica- lar myocytes (NRVMs) were isolated from 1 to 2-d-old tion (Leica software) for >100 cells in each treatment. Rela- Sprague-Dawley rats by enzymatic digestion with 0.1% tive rates of protein synthesis were determined trypsin (Hyclone) and 0.03% collagenase (Worthington by incuba- tion of myocytes with 2 µCi·mL-1 [3H] leucine (Amersham, Biochemical), as described in a previous study[16]. After in- UK) for 6 h and then incubated in 10% trichloroacetic acid cubating the cell-containing supernatant for 1.5 h to remove fibroblasts, cells were plated onto laminin-treated 35-mm for 30 min on ice to precipitate the protein. The precipitates were washed twice with cold water and then were solubilized dishes at a density of 1.0×103−1.2×103 cells/mm2 unless speci- in 0.1% SDS+0.3 mol·L-1 o 3 fied otherwise. The culture medium, Dulbecco’s modified NaOH at 37 C for 1 h. [ H]leucine incorporation was then quantified for radioactivity (Beckman Eagle’s medium (DMEM) and Medium 199 (4:1) containing LS 1801) from triplicate aliquots of each sample. 10% fetal bovine serum, 4 mmol·L-1 L-glutamine, 100 U/mL Materials 2-Aminoethoxydiphenylborate (2-APB) and penicillin and streptomycin, and 0.1 mmol·L-1 5-bromo-2- xestospongin C (Xe C) were from Calbiochem. Ryanodine deoxyuridine (Roche Molecular Biochemicals), was replaced and phenylephrine (PE) were purchased from Sigma-Aldrich. at 42 h with serum-free medium, and cells were further cul- Myo-inositol 1,4,5-trisphosphate hexakis (butyryloxymethyl) tured for 6 h. Cultured cells exhibited >95% positive staining ester (IP BM) was synthesized as described in a previous for α-actinin, and began spontaneous contraction 24–48 h 3 study (purity >95%)[17]. into culture. Statistical analysis The data were analyzed and pre- Reverse transcription-polymerase chain reaction Myoc sented as mean±SEM. When appropriate, statistical com- ytes were lysed with TRIzol reagent (Invitrogen) and clari- parison was carried out with two-way paired or unpaired fied by centrifugation. Analysis of mRNA levels for atrial natriuretic factor (ANF) and 18 s were performed with prim- Student’s t-test. The accepted level of significance was P<0.05. ers designed to detect rat gene products. ANF detection used primers 5'-TCCCAGGCCATATTGGAGCA-3' and 5'- Results CAGCGAGAGCCCTCAGT-3', generating a 306 bp fragm ent. 2+ Detection of mRNA 18 s used primers 5'-TGCAGCCCCGGA- IP3BM-induced potentiation of Ca oscillations In a pre- CATCTAAG-3' and 5'-GGAAGGGCACCACCAGGAGT-3', vious study (submitted to PNAS) we identified that IP3 for- generating a 317 bp fragment. RT-PCR reactions were per- mation and spontaneous global Ca2+ transients were in- α formed with 2 µg of total RNA and followed by 30 cycles of creased in a dose-dependent manner after 1AR stimulation 896 Http://www.chinaphar.com Luo DL et al with PE. Additionally, local Ca2+ sparks and waves, espe- Pretreatment of cells with 2-APB 4 µmol·L-1 or Xe C 10 µmol·L-1 cially in the perinuclear area, were enhanced significantly for 10 min robustly inhibited IP3BM-induced potentiation of upon PE stimulation, concomitant with the enriched distri- Ca2+ oscillations, whereas ryanodine, a RyRs inhibitor, at a -1 bution of IP3Rs in this area. More importantly, inhibition of concentration of 30 µmol·L failed to block the IP3BM effect − IP3Rs with membrane permeable IP3R inhibitors, 2-APB and (Figures 1B D).