742 Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748 http://www.actaps.com.cn Research Paper Cardiac hypertrophy induced by prostaglandin F2α may be mediated by calcineurin signal transduction pathway in rats JIANG Qing-Song1, 2 , HUANG Xie-Nan1,*, YANG Gui-Zhong1, 2, DAI Zhi-Kai1, ZHOU Qi-Xin2 , SHI Jing-Shan1, WU Qin1 1 Department of Pharmacology, Zunyi Medical College, Zunyi 563003, China; 2 Department of Pharmacology, Chongqing Medical University, Chongqing 400016, China Abstract: In this paper, we studied the relationship between the prostaglandin F2α (PGF2α)-induced cardiac hypertrophy and calcineurin (CaN) signal transduction pathway in vivo and in vitro. Male Sprague-Dawley rats were given a single i.p. injection with monocrotaline (MCT) (60 mg/kg) and then given orally with celecoxib (20 mg/kg) or vehicle once a day for 14 d before (from d 1 to d 14) or after (from d 15 to d 28) right ventricular hypertrophy (RVH) was formed. Body weight (BW), right ventricular weight (RV), left ventricular with septum weight (LV), as well as lung weight were determined. RVH index (RVHI=RV/LV), RV/BW, and lung weight/BW were calculated and histological changes were observed with transmission electron microscope. PGF2α level, atrial natriuretic peptide (ANP) and CaN mRNA expressions, expression of CaN and its downstream effectors, NFAT 3 and GATA4 protein were assayed by EIA kit, RT-PCR, and Western blotting, respectively. The cardiomyocyte hypertrophy in primary culture induced by PGF2α (0.1 µmol/L) was evaluated by measuring the cell diameter, protein content, and ANP mRNA as well as CaN mRNA expressions. It was found that 14 d or 28 d after MCT was given, the RVHI, RV/BW, and lung weight/BW were significantly increased by 47%, 53% and 118%, and by 64%, 94% and 156%, respectively; at the same time PGF2α levels in RV tissue were increased by 44% and by 51% with increasing RVHI, and elevated expressions of ANP and CaN mRNA, as well as CaN, NFAT 3 and GATA4 proteins in a positive correlation manner. Furthermore, some histological injuries were found in RV tissue. Celecoxib, a cyclooxygenase inhibitor, obviously blunted the elevation of RVHI, RV/BW, and lung weight/BW no matter it was given before or after RVH. In vitro experiments showed that 0.1 µmol/L PGF2α significantly increased the cardiomyocyte diameter and protein content, and promoted ANP and CaN mRNA expressions, which was blocked by cyclosporin A, a CaN inhibitor. Our results indicate that PGF2α may be involved in cardiac hypertrophy induced by MCT in rats through CaN signal transduction pathway. Key words: prostaglandin F2α; cardiac hypertrophy; calcineurin; monocrotaline F 2α 1,2 1,* 1,2 1 2 1 1 1 2 563003 400016 (monocrotaline MCT) F2α (prostaglandin F2α PGF2α ) (calcineurin CaN) Sprague-Dawley MCT (60 mg/kg) i.p. (20 mg/kg) / 2 EIA PGF2α RT-PCR (atrial natriuretic peptide ANP) CaN mRNA CaN NFAT 3 GATA4 ANP mRNA 0.1 µmol/L PGF2α CaN mRNA CaN A PGF2α CaN mRNA MCT 2 (M2W ) (RVHI) / / 47% 53% 118% 4 (M4W ) Received 2005-03-30 Accepted 2005-08-15 This work was supported by the Science Foundation of Guizhou Province (No.2004-3057). * Corresponding author. Tel : +86-852-8205416; E-mail: firstname.lastname@example.org JIANG Qing-Song et al: Cardiac Hypertrophy Induced by PGF2α May be Mediated by CaN 743 64% 94% 156% PGF2α M2W M4W 44% 51% RVHI ANP CaN mRNA CaN/NFAT 3 /GATA4 MCT PGF2α (0.1 µmol/L) ANP CaN mRNA CaN A PGF2α CaN mRNA PGF2α MCT CaN F2α Q257; R542.2 It has been well known that cardiac hypertrophy, which is lates several downstream effectors such as calcineurin caused by chronic increase in cardiac workload, may be (CaN) and Ca 2+/calmodulin-dependent protein kinases beneficial for body to maintain normal cardiac function. (CaMKs). CaN is a key mediator of cardiac hypertrophy [10, 11]. On the other hand, hypertrophy definitely leads to dys- It dephosphorylates nuclear factor of activated T cells function of heart, even congestive heart failure, arrhyth- (NFAT), which then translocates to the nucleus where it mia and sudden death. Therefore, it is very important to acts combinatorially with other transcription factors (e.g. understand the precise mechanisms and mediators of car- GATA4) for activation of downstream targets to induce diac hypertrophy. cardiac hypertrophy [12,13]. However, whether PGF2α-in- It has been reported that many vasoactive substances duced cardiac hypertrophy is mediated by CaN-signal path- were implicated in cardiac and vascular remodeling, but way has not been known yet. only a few studies focused on prostaglandin F2α (PGF2α) In this study, we took right ventricular hypertrophy which caused cultured rat cardiomyocytes over hypertro- (RVH) model of rat induced by monocrotaline (MCT) phy and over expression of atrial natriuretic peptide (ANP). and cultured neonatal rat cardiomyocytes to investigate the Some data showed that experimental infarction, ischemia, possible relationship between CaN-signaling pathway and and pressure overload increased the formation of prostag- PGF2α in cardiac hypertrophy. landins (PGs) in the myocardium[3-5]. These findings indi- cate that PGF2α is a potential candidate to mediate the car- 1 MATERIALS AND METHODS diac hypertrophy. 1.1 Animals In general, PGF2α exerts its biological effects through FP Male Sprague-Dawley rats (200~250 g, Grade II, Certifi- prostanoid receptor, which is coupled with activation of cate No. SCXK Chongqing 20020003, China) were housed phospholipase C via Gq protein, leading to inositol triphos- in a standard environment with a 12 h light /12 h dark phate-induced elevation of intracellular calcium and cycle, where they were freely access to food and water. diacylglycerol activation of protein kinase C (PKC). One week after being fed adaptively, the rats were injected Stimulation of Gq protein-linked receptors can also lead to (i.p.) with MCT solution (Sigma Chemical Co., USA, 60 activation of members of the mitogen-activated protein ki- mg/kg, n=40) or equal volumes of vehicle (n=21). After nase (MAPK) family, including extracellular signal-regu- that, the animals were divided into 7 groups: (1) vehicle- lated kinase (ERK), p38 mitogen-activated protein kinase treated rats (3 groups): after vehicle was given, the rats (p38), and c-Jun NH2-terminal kinase (JNK), which are were given i.g. with vehicle once a day for 0, 2 or 4 weeks thought to play a critical role in cell proliferation and differ- (N0W, N2W, and N4W groups) (n=7), respectively; (2) entiation as well as cardiac hypertrophy. It was said that MCT-treated rats (2 groups): after MCT-treatment, the rats PGF2α-induced cardiomyocyte hypertrophy and ANP ex- were given i.g. with vehicle once a day for 2 or 4 weeks pression is independent of PKC, p38, or ERK pathways, (M2W or M4W groups) (n=10); (3) MCT plus celecoxib- therefore, the possible signal transduction pathway is quite treated rats (2 groups): after MCT-treatment, the rats were different from that of known activation of Gq. given i.g. with celecoxib (G.D. Searle & Co., USA) 20 Ca2+ signaling has been reported to play a critical role in mg/kg·d-1 for 14 d from d 1 to d 14 or from d 15 to d 28 the generation of cardiac hypertrophy induced by various (PC2W and TC2W groups) (n=10). hypertrophic stimuli, including angiotensin II, endothelin- MCT was prepared by dissolving in 1 mol/L HCl. The I, α-adrenergic agents and mechanical stress. The in- pH was neutralized with 0.5 mol/L NaOH, and final con- creased intracellular Ca2+ binds to calmodulin and regu- centration was adjusted to 2% with phosphate-buffered 744 Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748 saline (PBS, pH 7.4). AAG CTG TTG CAG CCT A -3’; (2) CaN: sense 5’- ACT 1.2 Measurement of hemodynamics GGC ATG CTC CCC AGC GGA -3’, antisense 5’- GTG The animals were weighed and anesthetized with chloral CCG TTA GTC TCT GAG GCG -3’; (3) β-actin: sense hydrate solution (0.4 g/kg, i.p.), and then the carotid ar- 5’- GAC TAC CTC ATG AAG ATC CTG ACC -3’, antisense tery was dissociated so as to insert the artery cannulae to 5’- TGA TCT TCA TGG TGC TAG GAG CC -3’; the measure systolic pressure/diastolic pressure (SP/DP), mean predicted product size were 202 bp, 244 bp, and 423 bp, arterial pressure (AP) and heart rate (HR). respectively. These primers were synthesized by Beijing 1.3 Determination of heart weights Dingguo Shengwu Jishu Fazhan Ltd. Co. (China). RT-PCR After measurement of hemodynamics, the rats were killed was performed according to the RT-PCR Kit protocols by decapitation, and then the lung and heart were removed (Promega, USA) in a DNA thermal cycler (PCR instrument, and weighed quickly. The heart was separated into the Bio-Rad, USA). Cycling conditions were: (1) 1 cycle of right ventricle (RV) and the left ventricle with septum (LV) 48 °C for 45 min, 94 °C for 2 min; (2) 35 cycles of 94 °C and weighed separately. Finally, the lung weight/body for 30 s, 60 °C for 30 s, and 72 °C for 1 min; (3) 72 °C for weight (BW), RV hypertrophic index (RVHI=RV/LV) and 7 min. The products were electrophoresed on a 1% agar- RV/BW were calculated. After weighing, the RV tissue was ose gel and visualized with ethidium bromide. The optical quickly frozen and kept at –70 °C until PGF2α, protein or density values (OD) for each band of ANP, CaN and β- total RNA were isolated. actin on the gel were assayed by BI2000 Imaging Analysis 1.4 Histological observation System (Chengdu Taimeng Sci-Tec Ltd. Co., China). β- After RV walls (1 mm3) were fixed by immersion in cold actin was used as an internal control for semi-quantitative 2.5% glutaraldehyde solution (pH 7.4), they were rinsed assay. and postfixed with 1% osmium tetroxide in 0.1 mol/L PBS 1.7 Western blotting for RV tissue proteins for 2 h at room temperature, and then dehydrated through Forty microgrammes of protein from the supernatant of a graded series of ethanol to propylene oxide and embed- homogenized rat RV tissues centrifuged at 12 000 g was ded in epoxy resin. 60 nm section in thick were made and separated by 10% SDS-PAGE and transferred on then observed with transmission electron microscope. polyvinylidene difluoride nylon membranes. Protein con- 1.5 Myocyte PGF2α isolation and measurements centrations were determined by the Bradford assay us- Three hearts per group were collected for myocyte isola- ing bovine γ-globulin as standard. The blots were probed tion using the methods described by Zhang  and PGF2α with mouse anti-CaNA-α (1:200 dilution) or anti-NFAT3 contents were measured with EIA kit (Cayman Chemical (1:100 dilution) or anti-GATA4 antibodies (1:100 dilution) Co., USA). In brief, frozen tissues (30 mg) were homog- (Santa Cruz Biotechnology, USA) followed by horseradish enized immediately in 1.0 ml absolute ethanol and normal peroxidase-conjugated goat anti-mouse IgG (1:2 500 saline (2:8) using a motor-driven glass homogenizer. After dilution) antibodies. Immunodetection was carried out us- added with HCl 50 µl at 0.1 mol/L and ethyl acetate 5.0 ml, ing BI2000 Imaging Analysis System. the homongenate was centrifuged at 2 500 g for 20 min, 1.8 Cell culture then the supernatant was lyophilized with nitrogen and Neonatal rat ventricular myocytes from 1- to 3-day-old stored at –70 °C. For the measurement of PGF2α, the lyo- Sprague-Dawley rats were prepared and cultured for 48 h philized residues were dissolved in 0.5 ml HEPES and its in Dulbecco’s modified Eagle’s medium (DMEM) con- absorption were assayed at 405 nm. The PGF2α content of taining 20% fetal bovine serum and bromodeoxyuridine each sample was calculated by the standard curve. (0.1 mmol/L) as described previously. The media was 1.6 RNA extraction and RT-PCR replaced by serum-free DMEM for further 24 h before Total RNA extracted from right ventricles according to the treatments. PGF2α (0.1 µmol/L) was used to stimulate neo- RNeasy Mini Handbook protocol (Qiagen Company, natal rat cardiomyocytes, and cyclosporin A, a CaN Germany) was redissolved in 30 to 50 µl of DEPC water, inhibitor, was used to investigate the relation between the and stored at –70 °C. RNA was spectrophotometrically myocyte hypertrophy induced by PGF2α and CaN signal quantified by measuring the optical density of samples at pathway. Cell diameter was measured by BI2000 Imaging 260/280 nm. Analysis System. Protein concentration and ANP mRNA The nucleotide sequence of the primers used in this ex- as well as CaN mRNA expressions were determined by periment were as follows[16, 17]: (1) ANP: sense 5’- GCC the methods described above. CTG AGC GAG CAG ACC GA -3’, antisense 5’- CGG 1.9 Statistical analysis JIANG Qing-Song et al: Cardiac Hypertrophy Induced by PGF2α May be Mediated by CaN 745 All of the data were expressed as mean ± SD. The data 2.3 Myocardial morphology were analyzed statistically using the SPSS 11.5 for Win- Pathomorphology of the RV was observed with electron dows statistical program by one-way ANOVA or Student’s microscopy. It was shown that the hypertrophied RV tis- t-test. Correlation analyses were used for observing the sues from MCT-treated animals showed some injuries: dis- correlation between PGF2α level with cardiac hypertrophy ruption of the intercalated disk, irregular pattern of cross- or with CaN signaling pathway. Differences were consid- striation, and aggregation of swollen and misshapen mito- ered statistically significant when P value was less than chondria (Fig. 1). 0.05. 2.4 Levels of PGF2α The calculating 50% B/B0 was 41 pg/ml and the detection 2 RESULTS limit (80% B/B0) was 9 pg/ml using standard curve. The PGF2α content was (6.30 ± 0.50) pg/mg tissue in N0W. 2.1 Hemodynamics No changes were found in N2W and N4W [(6.03 ± 0.59), The HR, SP/DP and AP were (351 ± 28) bpm and (136 ± (6.53 ± 0.29) pg/mg tissue, respectively]. But in MCT- 12), (108 ± 8), (117 ± 10) mmHg, respectively, in N0W treated rats, the PGF2α contents significantly increased by group. No changes were found in other groups, including 44% and 51% in M2W and M4W, respectively (P<0.05, N2W, N4W, M2W, M4W, PC2W and TC2W groups (data n=6) (Fig.2), which was related to RVHI with correlation not shown). coefficient of 0.75 (P<0.001). 2.2 RV tissue weights 2.5 Expressions of ANP and CaN mRNA Table 1 showed that the BW in vehicle-treated group (N0W, N2W and N4W) increased in time-dependent manner, but Expressions of the cardiac ANP and CaN mRNA from RV in M2W and M4W decreased by 16% and 21%, tissues were shown in Table 2. It was obvious that there respectively, compared with that in N2W and N4W. The were no significant changes among N0W, N2W, and N4W RVHI, RV/BW, and lung weight/BW increased by 47%, groups. However, in M2W and M4W groups, ANP mRNA 53%, and 118% in M2W and 64%, 94%, and 156% in and CaN mRNA expressions distinctly increased in time- M4W, in comparison with corresponding control. Pretreat- dependent manner and were related to PGF2α levels with ment with celecoxib could significantly blunt the decrease correlation coefficients of 0.92 and 0.86 (P<0.001), of BW and the increase of RVHI, RV/BW and lung/BW respectively. (PC2W vs M2W). Although the BW in TC2W group had 2.6 CaN, NFAT3, GATA4 protein levels no change, its RVHI, RV/BW and lung weight/BW were Figure 3 showed that the CaN, NFAT3, and GATA4 protein obviously decreased by 9%, 18%, and 17%, respectively, levels were increased by 28%, 24%, and 15% in M2W when compared with that in M4W. (P<0.05), 44%, 28% and 21% in M4W (P<0.05), Table 1. General pathomorphological changes of heart and lung in rats treated with vehicle or monocrotaline or celecoxib + monocrotaline Groups BW (g) RVHI (g/g) RV/BW ( 10-4, g/g) Lung weight/BW ( 10-4, g/g) N0W 214 ± 13 0.2768 ± 0.0196 5.41 ± 0.41 51.98 ± 5.33 *** N2W 249 ± 21 0.2807 ± 0.0323 5.79 ± 0.52 52.39 ± 5.86 N4W 271 ± 22 ***# 0.2831 ± 0.0238 5.77 ± 0.65 53.30 ± 12.28 M2W 210 ± 23 ## 0.4113 ± 0.0470 ***### 8.86 ± 1.22 ***### 114.15 ± 16.36 ***### M4W 215 ± 16 +++ 0.4638 ± 0.0537 ***+++ 11.17 ± 1.42 ***+++ 136.42 ± 21.44***+++ * **## ***### PC2W 231 ± 16 0.3485 ± 0.0501 7.14 ± 0.60 89.91 ± 10.17***### TC2W 219 ± 21+++ 0.4209 ± 0.0195 ***+++ 9.18 ± 0.83 ***+++ 112.76 ± 17.84***+++ mean SD, ncontrol=7, nMCT=10. Groups: male Sprague-Dawley rats were injected (i.p.) with monocrotaline (60 mg/kg) and then given orally with vehicle for 2 weeks (M2W) or 4 weeks (M4W), or with celecoxib (20 mg/kg) from d 1 to d 14 (PC2W) or from d 15 to d 28 (TC2W) after monocrotaline was given. Control groups were injected (i.p.) with vehicle and then given orally with vehicle for 0, 2, or 4 weeks (N0W, N2W, N4W). BW, body weight; RVHI, right ventricular hypertrophy index; RV, right ventricular weight. *P<0.05, **P<0.5, *** P<0.001 vs N0W; #P<0.05, ##P<0.01, ###P<0.001 vs N2W; +++P<0.001 vs N4W; P<0.05 P<0.01, P<0.001 vs M2W; P<0.05, P<0.01 vs M4W. 746 Acta Physiologica Sinica, December 25, 2005, 57 (6): 742-748 Fig. 1. Electron microscopic findings of the right ventricular wall of the rats treated with vehicle (A) or with monocrotaline and then fed for 2 weeks (B). A did not show any abnormal changes, but B showed some myocardial injuries such as disruption of the intercalated disk (arrow), an irregular pattern of cross-striation, and an aggregation of swollen and misshapen mitochondria (M). Scale bar, 2.2 µm. Table 2. Expressions of ANP or CaN mRNA in right ventricle of rats treated with vehicle or monocrotalline Groups ANP mRNA (OD) CaN mRNA (OD) N0W 0.010 ± 0.002 0.226 ± 0.023 N2W 0.007 ± 0.003 0.227 ± 0.013 N4W 0.008 ± 0.001 0.227 ± 0.020 *** M2W 0.025 ± 0.002 0.394 ± 0.016** ### M4W 0.026 ± 0.003 0.424 ± 0.033## Fig. 2. PGF2α levels in right ventricle of rats treated with vehicle or mean ± SD, n =3. ANP, atrial natriuretic peptide; CaN, calcineurin. monocrotaline. Groups: male Sprague-Dawley rats were injected Groups: male Sprague-Dawley rats were injected (i.p.) with (i.p.) with monocrotaline (60 mg/kg) and then given orally with ve- monocrotaline (60 mg/kg) and then given orally with vehicle for 2 hicle for 2 weeks (M2W) or 4 weeks (M4W). Control groups were weeks (M2W) or 4 weeks (M4W). Control groups were injected injected (i.p.) with vehicle and then given orally with vehicle for 0, 2, (i.p.) with vehicle and then given orally with vehicle for 0, 2, or 4 or 4 weeks (N0W, N2W, N4W). *P<0.05 vs corresponding control. weeks (N0W, N2W, N4W). **P<0.01, ***P<0.001 vs N2W; ##P< 0.01, ###P<0.001 vs N4W. respectively, compared with corresponding controls. The correlation analysis of them with PGF2α levels showed that the correlation coefficients were 0.58, 0.57 and 0.63 (P<0.05), respectively. 2.7 Effect of cyclosporin A on cardiomyocyte hyper- trophy induced by PGF2α Table 3 showed that the treatment of PGF2α (0.1 µmol/L) Fig. 3. Expression of CaN, NFAT3 or GATA4 in right ventricle of significantly increased the diameter, protein content and rats treated with vehicle (N0W) or monocrotalline (60 mg/kg) i.p. and ANP mRNA expressin of the primary cultured then fed for 2 weeks (M2W) or 4 weeks (M4W) by Western blotting cardiomyocytes and cyclosporin A (1 µmol/L) significantly (OD). *P<0.05, **P<0.01 vs N0W. decreased the elevation (P<0.01). Similar result was found JIANG Qing-Song et al: Cardiac Hypertrophy Induced by PGF2α May be Mediated by CaN 747 Table 3. Effects of cyclosporin A on the increasing cardiaomyocyte diamter, protein content, and ANP mRNA and CaN mRNA expressions induced by PGF2α (mean ± SD) Groups (µmol/L) Cardiomyocyte Protein content ANP mRNA CaN mRNA diameter (µm) (n=3) (pg/cell)(n=6) expression (OD) (n=3) expression (OD) (n=3) Control 36 ± 11 419.2 ± 41.6 0.010 ± 0.004 0.225 ± 0.004 *** *** *** PGF2α (0.1) 115 ± 23 548.5 ± 59.2 0.026 ± 0.004 0.343 ± 0.030*** ### ## ## Cyclosporin A (1)+PGF2α (0.1) 67 ± 15 420.5 ± 69.7 0.019 ± 0.001 0.252 ± 0.033## ANP, atrial natriuretic peptide; CaN, calcineurin. ***P<0.001 vs control; ##P<0.01, ###P<0.001 vs PGF2α (0.1 µmol/L ) treatment. in the CaN mRNA expression of cardiomyocytes treated phy of cultured cardiac myocytes, it was considered that by PGF2α and interfered by cyclosporin A. PGF2α might participate in the process of RVH caused by MCT. Similar report from Lai et al. supports our opinion. 3 DISCUSSION Many molecular mechanisms are involved in the pro- cess of cardiac hypertrophy, including the factors and their Current data for identification of factors that induce car- interactions in the MAPK and PKC pathways. Recently, diac hypertrophy overwhelmingly derived from animal LV the Ca2+/calmodulin- activated CaN signaling pathway was hypertrophy models, such as spontaneously hypertensive also described as an important pathway in Gq-coupled re- rats and aortic banding in normotensive Sprague-Dawley ceptor induced hypertrophy[23, 24]. In MCT-induced hyper- rats, or from cultured cardiac myocyte in vitro. In the trophic model, we observed that there existed significant present study, we used MCT-induced RVH rats to probe parallel elevations about CaN mRNA expression, CaN, the possible role of PGF2α in the pathogenesis of RVH and NFAT3, GATA4 protein expressions, and PGF2α contents its signal transduction pathway because it has been well in RV tissue. However, in order to determine whether this known that MCT is converted in the liver to MCT-pyrrole hypertrophic model is related to CaN signal pathway and which causes injury of the lung vasculature leading to pul- PGF2α, it is necessary to observe if the CaN inhibitor de- monary hypertension and subsequently to RVH. In our presses the cardiac hypertrophy and CaN mRNA expre- study, the significant increase lung weight/BW ratio, RVHI, ssion. Because current CaN inhibitors have so many con- and RV/BW ratio without changes of blood pressure and founding effects that are associated with systemic admin- HR strongly suggest that there exists the formation of pul- istration of the drugs and make results confused, it is sel- monary hypertension and RVH without LV hypertrophy dom to use them as tools for testing the CaN-pathway in (LVH) in MCT-treated rats, which meant that the estab- pressure-overload hypertrophy in vivo. For this reason, lishment of RVH model was successful. Furthermore, the we took the myocyte hypertrophy model caused by PGF2α observation that the changes of myocardial pathomorphol- in vitro and found cyclosporin A, a CaN inhibitor, obvi- ogy were similar to the description of RVH with increasing ously blocked the myocyte hypertrophy process and CaN expression of ANP mRNA in MCT-treated rats provided mRNA expression. 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